Investigation of the enzymatic processes depend on type of reaction, due to the classification of enzymes. Units of enzyme activity, regulation of enzymatic processes and
mechanism of enzymopathy appearance.
The principle of international
classification and nomenclature of enzymes.
The first general principle of
these 'Recommendations' is that names purporting to be names of enzymes,
especially those ending in -ase, should be used
only for single enzymes, i.e. single catalytic entities. They should not
be applied to systems containing more than one enzyme. When it is desired to
name such a system on the basis of the overall reaction catalysed by it, the
word system should be included in the name. For example, the system
catalysing the oxidation of succinate by molecular oxygen, consisting of succinate dehydrogenase, cytochrome oxidase, and several
intermediate carriers, should not be named succinate oxidase, but it may
be called the succinate oxidase system. Other examples of systems
consisting of several structurally and functionally linked enzymes (and
cofactors) are the pyruvate dehydrogenase system, the similar 2-oxoglutarate
dehydrogenase system, and the fatty acid synthase system.
In this context it is appropriate to express
disapproval of a loose and misleading practice that is found in the biological
literature. It consists in designation of a natural substance (or even of an
hypothetical active principle), responsible for a physiological or biophysical
phenomenon that cannot be described in terms of a definite chemical reaction,
by the name of the phenomenon in conjugation with the suffix -ase, which
implies an individual enzyme. Some examples of such phenomenase
nomenclature, which should be discouraged even if there are reasons to suppose
that the particular agent may have enzymic properties, are: permease,
translocase, reparase, joinase, replicase, codase, etc..
The second general principle
is that enzymes are principally classified and named according to the reaction
they catalyse. The chemical reaction catalysed is the specific property that
distinguishes one enzyme from another, and it is logical to use it as the basis
for the classification and naming of enzymes.
Several alternative bases for classification and
naming had been considered, e.g. chemical nature of the enzymes (whether
it is a flavoprotein, a hemoprotein, a pyridoxal-phosphate protein, a copper
protein, and so on), or chemical nature of the substrate (nucleotides,
carbohydrates, proteins, etc.). The first cannot serve as a general
basis, for only a minority of enzymes have such
identifiable prosthetic groups. The chemical nature of the enzyme has, however,
been used exceptionally in certain cases where classification based on
specificity is difficult, for example, with the peptidases (subclass EC 3.4). Thus, the
intimate mechanism of the reaction, and the formation of intermediate complexes
of the reactants with the enzyme is not taken into account, but only the
observed chemical change produced by the complete enzyme reaction. For example,
in those cases in which the enzyme contains a prosthetic group that serves to
catalyse transfer from a donor to an acceptor (e.g. flavin, biotin, or
pyridoxal-phosphate enzymes) the name of the prosthetic group is not normally
included in the name of the enzyme. Nevertheless, where alternative names are
possible, the mechanism may be taken into account in choosing between them.
A second consequence of this concept is that a certain
name designates not a single enzyme protein but a group of proteins with the
same catalytic property. Enzymes from different sources (various bacterial,
plant or animal species) are classified as one entry. Some are justified
because the mechanism of the reaction or the substrate specificity is so
different as to warrant different entries in the enzyme list. This applies, for
example, to the two cholinesterases, EC 3.1.1.7 and 3.1.1.8, the two citrate
hydro-lyases, EC 4.2.1.3 and 4.2.1.4, and the two amine oxidases, EC 1.4.3.4
and 1.4.3.6. Others are mainly historical, e.g. acid and alkaline
phosphatases (EC 3.1.3.1 and EC 3.1.3.2).
A third general principle
adopted is that the enzymes are divided into groups on the basis of the type of
reaction catalysed, and this, together with the name(s) of the substrate(s)
provides a basis for naming individual enzymes. It is also the basis for
classification and code numbers.
Special problems attend the classification and naming of
enzymes catalysing complicated transformations that can be resolved into
several sequential or coupled intermediary reactions of different types, all
catalysed by a single enzyme (not an enzyme system). Some of the steps may be
spontaneous non-catalytic reactions, while one or more intermediate steps
depend on catalysis by the enzyme. Wherever the nature and sequence of
intermediary reactions is known or can be presumed with confidence,
classification and naming of the enzyme should be based on the first
enzyme-catalysed step that is essential to the subsequent transformations,
which can be indicated by a supplementary term in parentheses, e.g.
acetyl-CoA:glyoxylate C-acetyltransferase (thioester-hydrolysing,
carboxymethyl-forming) (EC 2.3.3.9, cf. section 3).
To classify an enzyme according to the type of
reaction catalysed, it is occasionally necessary to choose between alternative
ways of regarding a given reaction. In general, that alternative should be
selected which fits in best with the general system of classification and
reduces the number of exceptions.
Common
and Systematic Names
The first Enzyme Commission gave much thought to the
question of a systematic and logical nomenclature for enzymes, and finally
recommended that there should be two nomenclatures for enzymes, one systematic,
and one working or trivial. The systematic name of an enzyme, formed in
accordance with definite rules, showed the action of an enzyme as exactly as
possible, thus identifying the enzyme precisely. The trivial name was
sufficiently short for general use, but not necessarily very systematic; in a
great many cases it was a name already in current use. The introduction of
(often cumbersome) systematic names was strongly criticised. In many cases the
reaction catalysed is not much longer than the systematic name and can serve
just as well for identification, especially in conjunction with the code
number.
The Commission for Revision of Enzyme Nomenclature
discussed this problem at length, and a change in emphasis was made. It was
decided to give the trivial names more prominence in the Enzyme List; they now
follow immediately after the code number, and are described as Common Name.
Also, in the index the common names are indicated by an asterisk. Nevertheless,
it was decided to retain the systematic names as the basis for classification
for the following reasons:
(i)
the code number alone is only useful for
identification of an enzyme when a copy of the Enzyme List is at hand, whereas
the systematic name is self-explanatory;
(ii)
the systematic name stresses the type of reaction, the
reaction equation does not;
(iii)
systematic names can be formed for new enzymes by the
discoverer, by application of the rules, but code numbers should not be
assigned by individuals;
(iv)
common names for new enzymes are frequently formed as
a condensed version of the systematic name; therefore, the systematic names are
helpful in finding common names that are in accordance with the general
pattern.
Scheme
for the classification of enzymes and the generation of EC numbers
The first Enzyme Commission, in its report in 1961,
devised a system for classification of enzymes that also serves as a basis for
assigning code numbers to them. These code numbers, prefixed by EC, which are
now widely in use, contain four elements separated by points, with the
following meaning:
(i) the first number shows to
which of the six main divisions (classes) the enzyme belongs,
(ii) the second figure
indicates the subclass,
(iii) the third figure gives
the sub-subclass,
(iv) the fourth figure is the
serial number of the enzyme in its sub-subclass.
Enzyme
Classifications
Currently enzymes are grouped into
six functional classes by the International Union of Biochemists
(I.U.B.).
Number |
Classification |
Biochemical Properties |
1. |
Oxidoreductases |
Act
on many chemical groupings to add or remove hydrogen atoms. |
2. |
Transferases |
Transfer
functional groups between donor and acceptor molecules. Kinases are specialized
transferases that regulate metabolism by transferring phosphate from ATP to
other molecules. |
3. |
Hydrolases |
Add
water across a bond, hydrolyzing it. |
4. |
Lyases |
Add
water, ammonia or carbon dioxide across double bonds, or remove these
elements to produce double bonds. |
5. |
Isomerases |
Carry
out many kinds of isomerization: L to D isomerizations, mutase reactions
(shifts of chemical groups) and others. |
6. |
Ligases |
Catalyze
reactions in which two chemical groups are joined (or ligated) with the use
of energy from ATP. |
These rules give each enzyme a unique number. The I.U.B. system also
specifies a textual name for each enzyme. The enzyme's name is comprised of the
names of the substrate(s), the product(s) and the enzyme's functional class.
Because many enzymes, such as alcohol dehydrogenase, are widely known in the
scientific community by their common names, the change to I.U.B.-approved
nomenclature has been slow. In everyday usage, most enzymes are still called by
their common name.
Enzymes are also classified on the basis of their
composition. Enzymes composed wholly of protein are known as simple enzymes in
contrast to complex enzymes, which are composed of protein plus a relatively
small organic molecule. Complex enzymes are also known as holoenzymes. In this
terminology the protein component is known as the apoenzyme, while the
non-protein component is known as the coenzyme or prosthetic group where
prosthetic group describes a complex in which the small organic molecule is
bound to the apoenzyme by covalent bonds; when the binding between the
apoenzyme and non-protein components is non-covalent, the small organic
molecule is called a coenzyme. Many prosthetic groups and coenzymes are
water-soluble derivatives of vitamins. It should be noted that the main
clinical symptoms of dietary vitamin insufficiency generally arise from the
malfunction of enzymes, which lack sufficient cofactors derived from vitamins
to maintain homeostasis.
The non-protein component of an enzyme may be as simple as a metal ion
or as complex as a small non-protein organic molecule. Enzymes that require a
metal in their composition are known as metalloenzymes if they bind and retain
their metal atom(s) under all conditions with very high affinity. Those which have a lower affinity for metal ion, but still require
the metal ion for activity, are known as metal-activated enzymes.
Hydrolases. Mechanism of action of esterases,
peptidases, glycosidases. Examples.
Hydrolases.
These enzymes catalyse the hydrolytic cleavage of C-O, C-N, C-C and some
other bonds, including phosphoric anhydride bonds. Although the systematic name
always includes hydrolase, the common name is, in many cases, formed by
the name of the substrate with the suffix -ase.
It is understood that the name of the substrate with this suffix means a
hydrolytic enzyme.
A number of hydrolases acting on ester, glycosyl, peptide,
amide or other bonds are known to catalyse not only hydrolytic removal of a
particular group from their substrates, but likewise the transfer of this group
to suitable acceptor molecules. In principle, all hydrolytic enzymes might be
classified as transferases, since hydrolysis itself can be regarded as transfer
of a specific group to water as the acceptor. Yet, in most cases, the reaction
with water as the acceptor was discovered earlier and is considered as the main
physiological function of the enzyme. This is why such enzymes are classified
as hydrolases rather than as transferases.
Some hydrolases (especially some of
the esterases and glycosidases) pose problems because they have a very wide
specificity and it is not easy to decide if two preparations described by
different authors (perhaps from different sources) have the same catalytic
properties, or if they should be listed under separate entries. An example is vitamin
A esterase (formerly EC 3.1.1.12, now believed to be identical with EC 3.1.1.1).
To some extent the choice must be arbitrary; however, separate entries should
be given only when the specificities are sufficiently different.
Another problem is that proteinases have 'esterolytic'
action; they usually hydrolyse ester bonds in appropriate substrates even more
rapidly than natural peptide bonds. In this case, classification among the
peptide hydrolases is based on historical priority and presumed physiological
function.
The second figure in the code number of the hydrolases
indicates the nature of the bond hydrolysed; EC 3.1 are the esterases;
EC 3.2 the glycosylases, and so on.
The third figure normally specifies the nature of the
substrate, e.g. in the esterases the carboxylic ester hydrolases
(EC 3.1.1), thiolester hydrolases (EC 3.1.2), phosphoric monoester
hydrolases (EC 3.1.3); in the glycosylases the O-glycosidases (EC
3.2.1), N-glycosylases (EC 3.2.2), etc. Exceptionally, in the
case of the peptidyl-peptide hydrolases the third figure is based on the
catalytic mechanism as shown by active centre studies or the effect of pH.
Esterases
- enzymes
that hydrolyse esters,
i.e. cleave the ester linkage to form free acid and alcohol.
Those that hydrolyse the ester linkages of fats are generally known as lipases, and those
that hydrolyse phospholipids
as phospholipases. Any enzymes catalyses the hydrolysis of an
ester.
Esterases form subclass EC 3.1 of the class EC 3,
hydrolases, and are divided according to the nature of their substrates into
the following sub-subclasses: EC 3.1.1, carboxylic ester hydrolases; EC 3.1.2,
thiolester hydrolases; EC 3.1.3, phosphatases, phosphoric monoester hydrolases;
EC 3.1.4, phosphodiesterases, phosphodiester hydrolases, phosphoric diester
hydrolases (other than nucleases; see EC 3.1.11 — 3.1.31 below); EC 3.1.5;
triphosphoric monoester hydrolases; EC 3.1.6, sulfatases, sulfuric ester
hydrolases; EC 3.1.7, pyrophosphatases, diphosphoric monoester hydrolases; EC
3.1.8, phosphoric triester hydrolases; EC 3.1.11, exodeoxyribonucleases
producing 5′-phosphomonoesters; EC 3.1.13, exoribonucleases producing 5′-phosphomonoesters;
EC 3.1.14, exoribonucleases producing other than 5′-phosphomonoesters; EC
3.1.15, exonucleases active with either ribo- or deoxyribonucleic acids and
producing 5′-phosphomonoesters; EC 3.1.16, exonucleases active with
either ribo- or deoxyribonucleic acids and producing other than 5′-phosphomonoesters;
EC 3.1.21, endodeoxyribonucleases producing 5′-phosphomonoesters; EC
3.1.22, endodeoxyribonucleases producing other than 5′-phosphomonoesters;
EC 3.1.25, endodeoxyribonucleases specific for altered bases; EC 3.1.26,
endoribonucleases producing 5′-phosphomonoesters; EC 3.1.27,
endoribonucleases producing other than 5′-phosphomonoesters; EC 3.1.30,
endonucleases active with either ribo- or deoxyribonucleic acids and producing
5′-phosphomonoesters; EC 3.1.31, endonucleases active with either ribo-
or deoxyribonucleic acids and producing other than 5′-phosphomonoesters.
Peptidase,
also called protease
or proteinase, is a type of enzyme that helps to break down proteins in the
body. This type of enzyme occurs naturally in the living things and forms part
of many metabolic processes. They form part of the larger systems in the body,
including the digestive, immune, and blood circulation systems. These enzymes
are classified into five different groups: aspartic proteinases, cysteine
proteinases, metalloproteinases, serine
proteinases, and threonine
proteases.
In
the digestive system, peptidases break down proteins by destroying the chains between
their amino acids,
and many can usually be found in the digestive tract. When protein enters the
body, it needs to be digested and broken down into smaller molecules so that it
can be used. This type of enzyme is responsible for this catabolic process.
Aspartic proteinases can usually be found in an acidic
environment like the stomach. They are responsible for the breakdown of food
and are also called pepsins. Other places that aspartic proteinases can be
found are in blood plasma
and in the immune system.
Glycoside hydrolases (also called
glycosidases or glycosyl hydrolases) catalyze
the hydrolysis
of the glycosidic linkage to release smaller sugars.
They are extremely common enzymes
with roles in nature including degradation of biomass
such as cellulose and hemicellulose,
in anti-bacterial defense strategies (e.g., lysozyme),
in pathogenesis
mechanisms (e.g., viral neuraminidases)
and in normal cellular function (e.g., trimming mannosidases
involved in N-linked glycoprotein biosynthesis).
Together with glycosyltransferases, glycosidases form the major
catalytic machinery for the synthesis and breakage of glycosidic bonds.
The
subclasses and sub-subclasses are formed according to principles indicated
below. he main divisions and subclasses are:
Oxidoreductases
Oxidoreductases are a class of enzymes
that catalyze oxidoreduction reactions. Oxidoreductases catalyze the
transfer of electrons from one molecule (the oxidant) to another molecule (the
reductant). Oxidoreductases catalyze reactions similar to the following,
A– + B → A + B– where A is the oxidant and B is the
reductant. Oxidorecuctases can be oxidases or dehydrogenases.
Oxidases are enzymes involved when molecular oxygen acts as an acceptor
of hydrogen or electrons. Whereas, dehydrogenases are enzymes that
oxidize a substrate by transferring hydrogen to an acceptor that is either NAD+/NADP+
or a flavin enzyme. Other oxidoreductases include peroxidases,
hydroxylases, oxygenases, and reductases. Peroxidases
are localized in peroxisomes, and catalyzes the reduction of hydrogen
peroxide. Hydroxylases add hydroxyl groups to its substrates.
Oxygenases incorporate oxygen from
molecular oxygen into organic substrates. Reductases catalyze reductions, in
most cases reductases can act like an oxidases.
Oxidoreductase enzymes play an important role in both
aerobic and anaerobic metabolism. They can be found in glycolysis, TCA
cycle, oxidative phosphorylation, and in amino acid metabolism. In
glycolysis, the enzyme glyceraldehydes-3-phosphate dehydrogenase catalyzes the
reduction of NAD+ to NADH. In order to maintain the re-dox
state of the cell, this NADH must be re-oxidized to NAD+, which
occurs in the oxidative phosphorylation pathway. Additional NADH
molecules are generated in the TCA cycle. The product of glycolysis,
pyruvate enters the TCA cycle in the form of acetyl-CoA. During anaerobic
glycolysis, the oxidation of NADH occurs through the reduction of pyruvate to
lactate. The lactate is then oxidized to pyruvate in muscle and liver
cells, and the pyruvate is further oxidized in the TCA cycle. All twenty
of the amino acids, except leucine and lysine, can be degraded to TCA cycle
intermediates. This allows the carbon skeletons of the amino acids to be
converted into oxaloacetate and subsequently into pyruvate. The
gluconeogenic pathway can then utilize the pyruvate forme
Transferases
Transferases are enzymes transferring a group, e.g. a methyl
group or a glycosyl group, from one compound (generally regarded as donor) to another
compound (generally regarded as acceptor). The systematic names are formed
according to the scheme donor:acceptor
grouptransferase. The common names are normally formed according to acceptor
grouptransferase or donor grouptransferase. In many cases, the donor
is a cofactor (coenzyme) charged with the group to be transferred. A special
case is that of the transaminases (see below).
Some
transferase reactions can be viewed in different ways. For example, the
enzyme-catalysed reaction X-Y + Z = X + Z-Y may be regarded either
as a transfer of the group Y from X to Z, or as a breaking of the X-Y bond by
the introduction of Z. Where Z represents phosphate or arsenate, the process is
often spoken of as 'phosphorolysis' or 'arsenolysis', respectively, and a
number of enzyme names based on the pattern of phosphorylase have come
into use. These names are not suitable for a systematic nomenclature, because
there is no reason to single out these particular enzymes from the other
transferases, and it is better to regard them simply as Y-transferases.
In
the above reaction, the group transferred is usually exchanged, at least
formally, for hydrogen, so that the equation could more strictly be written as:
X-Y
+ Z-H = X-H + Z-Y
Another
problem is posed in enzyme-catalysed transaminations, where the -NH2
group and -H are transferred to a compound containing
a carbonyl group in exchange for the =O of that group, according to the general
equation:
R1-CH(-NH2)-R2 + R3-CO-R4
R1-CO-R2 + R3-CH(-NH2)-R4
The reaction
can be considered formally as oxidative deamination of the donor (e.g.
amino acid) linked with reductive amination of the acceptor (e.g. oxo
acid), and the transaminating enzymes (pyridoxal-phosphate proteins) might be
classified as oxidoreductases. However, the unique distinctive feature of the
reaction is the transfer of the amino group (by a well-established mechanism
involving covalent substrate-coenzyme intermediates), which justified
allocation of these enzymes among the transferases as a special subclass (EC
2.6.1, transaminases).
Lyases
Lyases are enzymes cleaving C-C, C-O, C-N, and other bonds by
elimination, leaving double bonds or rings, or conversely adding groups to
double bonds. The systematic name is formed according to the pattern substrate
group-lyase. The hyphen is an important part of the name, and to avoid
confusion should not be omitted, e.g. hydro-lyase not 'hydrolyase'. In
the common names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2,
aldehyde, or water) are used. In cases where the reverse reaction is much more
important, or the only one demonstrated, synthase (not synthetase) may
be used in the name. Various subclasses of the lyases include
pyridoxal-phosphate enzymes that catalyse the elimination of a β-
or γ-substituent from an α-amino
acid followed by a replacement of this substituent by some other group. In the
overall replacement reaction, no unsaturated end-product is formed; therefore,
these enzymes might formally be classified as alkyl-transferases (EC
2.5.1...).
However, there is ample evidence that
the replacement is a two-step reaction involving the transient formation of
enzyme-bound α,β(or
β,γ)-unsaturated
amino acids. According to the rule that the first reaction is indicative for
classification, these enzymes are correctly classified as lyases.
Examples are tryptophan synthase (EC 4.2.1.20) and cystathionine β-synthase
(EC 4.2.1.22).
The second figure in the code number
indicates the bond broken: EC 4.1 are carbon-carbon
lyases, EC 4.2 carbon-oxygen lyases and so on.
The third figure gives further
information on the group eliminated (e.g. CO2 in EC 4.1.1, H2O
in EC 4.2.1).
Isomerases
These enzymes catalyse geometric or structural changes within one
molecule. According to the type of isomerism, they may be called racemases, epimerases,
cis-trans-isomerases, isomerases, tautomerases, mutases or
cycloisomerases.
In some cases, the interconversion in the substrate is
brought about by an intramolecular oxidoreduction (EC 5.3); since hydrogen
donor and acceptor are the same molecule, and no oxidized product appears, they
are not classified as oxidoreductases, even though they may contain firmly
bound NAD(P)+.
The subclasses are formed according to the type of
isomerism, the sub-subclasses to the type of substrates.
Ligases
Ligases are enzymes catalysing the
joining together of two molecules coupled with the hydrolysis of a diphosphate
bond in ATP or a similar triphosphate. The systematic names are formed on the
system X:Y ligase (ADP-forming). In earlier
editions of the list the term synthetase has been used for the common
names. Many authors have been confused by the use of the terms synthetase
(used only for Group 6) and synthase (used throughout the list when it
is desired to emphasis the synthetic nature of the reaction).
Consequently NC-IUB decided in 1983 to abandon the use
of synthetase for common names, and to replace them with names of the type X-Y
ligase. In a few cases in Group 6, where the reaction is more complex or
there is a common name for the product, a synthase name is used (e.g. EC
6.3.2.11 and EC 6.3.5.1).
It is recommended that if the term synthetase
is used by authors, it should continue to be restricted to the ligase group.
The second figure in the code number indicates the
bond formed: EC 6.1 for C-O bonds (enzymes acylating tRNA), EC 6.2 for C-S
bonds (acyl-CoA derivatives), etc. Sub-subclasses are only in use in the
C-N ligases.
In a few cases it is necessary to use the word other
in the description of subclasses and sub-subclasses. They have been
provisionally given the figure
http://www.youtube.com/watch?v=Ofs0mfkl370
Enzymes, specific to different organs. Localization of
enzymes in cell’s organells
An adaptive enzyme or inducible enzyme
is an enzyme
that is expressed only under conditions in which it is
clear of adaptive value, as opposed to a constitutive enzyme which is
produced all the time. The Inducible enzyme is used for the breaking-down of
things in the cell. It is also a part of the Operon Model, which illustrates a
way for genes to turn "on" and "off". The Inducer causes
the gene to turn on (controlled by the amount of reactant which turns the gene
on). Then there's the repressor protein that turns genes off.
The inducer can remove this repressor, turning genes back on. The
operator is a section of DNA where the repressor binds to shut off certain
genes; the promoter is the section of DNA where the RNA polymerase
binds. Lastly, the regulatory gene is the gene for the repressor protein. An
example of inducible enzyme is COX-2 which is synthesized in macrophages
to produce Prostaglandin E2 while the constitutive enzyme COX-1
(another isozyme in COX
family) is always produced in variety of organisms in body (like stomach).
Constitutive enzymes
are produced constitutively by the cell
under all physiological conditions.
Therefore, they are not controlled by induction or repression.
Constitutive enzymes are produced in
constant amounts without regard to the physiological demand or the
concentration of the substrate. They are continuously synthesized because their
role in maintaining cell processes or structure is indispensable.
The methods of separation and
purification of enzymes
Analysis
of the biological properties Understand its structure.
Study
interactions. No single procedure can be used to isolate every protein
Exploit
specific characteristics (structure or function) of the protein. Different
steps should exploit a different characteristic. Ensure method has little/no
effect on function.
Ammonium
sulphate precipitation (40%) exploits changes in the
solubility of proteins as consequence of a change in ionic strength (salt
conc.) of the solution
At
low salt, the solubility of a protein increases with salt concentration,
‘SALTING
IN’
But
as salt conc. (ionic strength) is increased further, the solubility of the
protein begins to decrease, until a point where the protein is precipitated
from solution,
‘SALTING
OUT’
Ion
Exchange Chromatography (IEC)
Separates
molecules based on their charge.
The
side-chain groups of some amino acids are ionizable, e.g., lysine, arginine,
histidine, glutamic acid, aspartic acid as are the N-terminal amino and
C-terminal carboxyl groups. Thus proteins are charge molecule s and can have a
different charge at a given pH because they have different compositions of
ionizable amino acids.
For
any given amphoteric protein, there will be a pH at which its overall charge is
0 (No. of negative charges equals the No. of positive charges).
This
is referred to as the ISOELECTRIC POINT (pI) or ISOTONIC POINT of the protein.
At a pH above its pI a protein will have a net negative charge while.
At
a pH below its pI a protein will have a net positive charge.
Gel
Filtration Chromatography (GFC)
GFC
(also Size Exclusion Chromatography, Molecular Sieve Chromatography or
Molecular Exclusion Chromatography)
Separates molecules based on their size (&
shape)
It
can also be used to determine the size and molecular weight of a protein
Separation
occurs due to the differential diffusion of various molecules into gel pores in
a porous matrix. For protein purification, the matrix typically consists of
porous beads (with pores of a specific size distribution) of an inert, highly
hydrated gel.
Separates
molecules based on specific interactions between the protein of interest and
the column matrix E.g. Antibodies which bind Protein. Enzyme which binds a
co-enzyme or inhibitor.
A
ligand is covalently bound to a solid matrix (usually agarose) which is then
packed into a chromatography column When a mixture containing the protein of
interest is applied to the column, the desired protein is bound by the
immobilised
ligands,
while all other proteins in the mixture, which should have no affinity for the
ligand pass through and are discarded
Affinity
chromatography (with HIS-tagged proteins).
Affinity
chromatography can be performed using a number of different protein tags.
poly-hisitidine.
The
histidine tag is very short (6 His residues).
Should
not alter the conformation of the tagged protein.
Should
not be involved in artificial interactions.
The
poly-his tag binds to a nickel chelate resin.
Eluted
by
PLASMA
ENZYMES
Measurements
of the activity of enzymes in plasma are of value in the diagnosis and
management of a wide variety of diseases. Most enzymes measured in plasma are
primarily intracellular, being released into the blood when there is damage to cell
membranes, but many enzymes, for example renin, complement factors and
coagulation factors, are actively secreted into the blood, where they fulfil
their physiological functions. Small amounts of intracellular enzymes are
present in the blood as a result of normal cell turnover. When damage to cells
occurs, increased amounts of enzymes will be released and their concentrations
in the blood will rise. However, such increases are not always due to tissue
damage. Other possible causes include:
· increased
cell turnover
· cellular
proliferation (e.g. neoplasia)
· increased
enzyme synthesis (enzyme induction)
· obstruction
to secretion
· decreased
clearance.
Little is known about the mechanisms by which enzymes
are removed from the circulation. Small molecules, such as amylase, are
filtered by the glomeruli but most enzymes are probably removed by
reticuloendothelial cells. Plasma amylase activity rises in acute renal failure
but, in general, changes in clearance rates are not known to be important as causes
of changes in plasma enzyme levels.
Plasma contains many functional enzymes, which
a actively secreted into plasma. For example, enzymes blood coagulation. On the
other hand, there are a few non functional enzymes in plasma, which are
coming out from cells of various tissues due to normal wear and tear. Their
normal levels in blood are very low; but are drastically increased during cell
death (necrosis) or disease. Therefore assays of these enzymes are very useful
in diagnosis diseases.
Enzyme
assays usually depend on the measurement the catalytic activity of the enzyme,
rather than the concentration of the enzyme protein itself. Since each enzyme
molecule can catalyze the reaction of many molecules of substrate, measurement
of activity provides great sensitivity. It is, however, important that the
conditions of the assay are optimized and standardized to give reliable and
reproducible results. Reference ranges for plasma enzymes are dependent on
assay conditions, for example temperature, and may also be subject to
physiological influences. It is thus important to be aware of both the
reference range for the laboratory providing the assay and the physiological
circumstances when interpreting the results of enzyme assays.
One international
unit is the amount of enzyme that will convert one
micromole of substrate per minute per litre of sample and is abbreviated as
U/L. The SI Unit (System Internationale) expression is more scientific, where
or Katal (catalytic activity) is defined as the number of mole of
substrate transformed per second per litre of sample. Katal is abbreviated as
kat or k (60 U = 1 μkat and 1 nk = 0.06 U).
A major disadvantage in the use of enzymes for the
diagnosis of tissue damage is their lack of specificity to a particular tissue
or cell type. Many enzymes are common to more than one tissue, with the result
that an increase in the plasma activity of a particular enzyme could reflect
damage to any one of these tissues. This problem may be obviated to some extent
in two ways:
first, different
tissues may contain (and thus release when they are damaged) two or more
enzymes in different proportions; thus alanine and aspartate aminotransferases
are both present in cardiac and skeletal muscle and hepatocytes, but there is
only a very little alanine aminotransferase in either type of muscle;
second,
some enzymes exist in different forms (isoforms), colloquially termed
isoenzymes (although, strictly, the term 'isoenzyme' refers only to a
genetically determined isoform). Individual isoforms are often characteristic
of a particular tissue: although they may have similar catalytic activities,
they often differ in some other measurable property, such as heat stability or
sensitivity to inhibitors.
After
a single insult to a tissue, the activity of intracellular enzymes in the
plasma rises as they are released from the damaged cells, and then falls as the
enzymes are cleared. It is thus important to consider the time at which the
blood sample is taken in relation to the insult. If taken too soon, there may
have been insufficient time for the enzyme to reach the blood- stream and if
too late, it may have been completely cleared. As with all diagnostic
techniques, data acquired from measurements of enzymes in plasma must always be
assessed in the light of whatever clinical and other information is available,
and their limitations borne in mind.
Most catalases exist as tetramers of 60 or 75 kDa, each subunit containing an active
site haem group buried deep within the structure, but which is accessible from
the surface through hydrophobic channels. The very rigid, stable
structure of catalases is resistant to unfolding, which makes them uniquely
stable enzymes that are more resistant to pH, thermal denaturation and
proteolysis than most other enzymes. Their stability and resistance to
proteolysis is an evolutionary advantage, especially since they are produced
during the stationary phase of cell growth when levels of proteases are high
and there is a rapid rate of protein turnover.
Haem-containing catalases break down hydrogen
peroxide by a two-stage mechanism in which hydrogen peroxide alternately
oxidises and reduces the haem iron at the active site. In the first step,
one hydrogen peroxide molecule oxidises the haem to an oxyferryl species.
In the second step, a second hydrogen peroxide molecule is used as a reductant
to regenerate the enzyme, producing water and oxygen. Some catalases
contain NADPH as a cofactor, which functions to prevent the formation of an
inactive compound. Catalases may have another role: the generation of
ROS, possibly hydroperoxides, upon UVB irradiation. In this way, UVB
light can be detoxified through the generation of hydrogen peroxide, which can
then be degraded by the catalase. NADPH may play a role in providing the
electrons needed to reduce molecular oxygen in the production of ROS.
Much of the hydrogen peroxide that is produced
during oxidative cellular metabolism comes from the breakdown of one of the
most damaging ROS, namely the superoxide anion radical (O2-).
Superoxide is broken down by superoxide dismutases into hydrogen peroxide and
oxygen. Superoxide is so damaging to cells that mutations in the superoxide
dismutase enzyme can lead to ALS, which is characterised by the loss of
motoneurons in the spinal cord and brain stem, possibly involving the
activation of caspase-12 and the apoptosis cascade via oxidative stress.
Regulation
of Antioxidant Enzymes
Antioxidant enzymes, including catalase, form the first line of defence against
free radicals, therefore their regulation depends mainly upon the oxidant
status of the cell.
However, there are other factors involved in their regulation, including
the enzyme-modulating action of various hormones such as growth hormone,
prolactin and melatonin. Melatonin is a derivative of the amino acid
tryptophan that acts as a neurohormone in mammals, but is also synthesized by
many other species, including plants, algae and bacteria. Melatonin has
been shown to markedly protect both membrane lipids and nuclear DNA from
oxidative damage. Melatonin can directly neutralise several ROS,
including hydrogen peroxide. It can also stimulate various antioxidant
enzymes, including catalase, either by increasing their activity or by
stimulating gene expression for these enzymes. The decrease in melatonin
levels observed with age correlates with an increase in neurogenerative
disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s
disease and stroke, all of which may involve oxidative stress. In
general, the production of ROS increases with aging and is associated with DNA
damage to the tissues.
By contrast, growth hormone, and possibly prolactin, was found to decrease
catalase and other antioxidant enzymes in various tissues in mice, suggesting
that this hormone acts as a suppressor of key antioxidant components.
The
origin of blood enzymes
Do you mean a blood enzymes test, or more generally,
enzymes in the blood?
Enzymes are proteins that carry out
chemical reactions (as opposed to structural enzymes). Most of the detectable
enzymes in the blood come from the various tissues and organs of the body.
Abnormal levels may reflect problems with a particular organ.
The most common blood enzymes test is
for liver enzymes. When the cells of the liver are damaged, such as from a
viral infection, their enzymes can leak out and be detected in the blood. Another
common test measures enzymes from heart damage, such as from a heart attack.
http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related
The
measurement of the serum levels of numerous enzymes has been shown to be of
diagnostic significance. This is because the presence of these enzymes in the
serum indicates that tissue or cellular damage has occurred resulting in the
release of intracellular components into the blood. Hence, when a physician
indicates that he/she is going to assay for liver enzymes, the purpose is to
ascertain the potential for liver cell damage.
Commonly assayed enzymes are the amino
transferases: alanine transaminase, ALT (sometimes still referred to as serum
glutamate-pyruvate aminotransferase, SGPT) and aspartate aminotransferase, AST
(also referred to as serum glutamate-oxaloacetate aminotransferase, SGOT);
lactate dehydrogenase, LDH; creatine kinase, CK (also called creatine
phosphokinase, CPK); gamma-glutamyl transpeptidase, GGT. Other enzymes are
assayed under a variety of different clinical situations but they will not be
covered here.
The typical liver enzymes measured are AST and
ALT. ALT is particularly diagnostic of liver involvement as this enzyme is
found predominantly in hepatocytes. When assaying for both ALT and AST the
ratio of the level of these two enzymes can also be diagnostic. Normally in
liver disease or damage that is not of viral origin the ratio of ALT/AST is
less than 1. However, with viral hepatitis the ALT/AST ratio will be greater
than 1. Measurement of AST is useful not only for liver involvement but also
for heart disease or damage. The level of AST elevation in the serum is
directly proportional to the number of cells involved as well as on the time
following injury that the AST assay was performed. Following injury, levels of
AST rise within 8 hours and peak 24-36 hours later. Within 3-7 days the level
of AST should return to pre-injury levels, provided a continuous insult is not
present or further injury occurs. Although measurement of AST is not, in and of
itself, diagnostic for myocardial infarction, taken together with LDH and CK
measurements (see below) the level of AST is useful for timing of the infarct.
The measurement of LDH is especially diagnostic
for myocardial infarction because this enzyme exist in 5 closely related, but
slightly different forms (isozymes). The 5 types and their normal distribution
and levels in non-disease/injury are listed below.
LDH 1 - Found in heart and
red-blood cells and is 17% - 27% of the normal serum total.
LDH 2 - Found in heart and
red-blood cells and is 27% - 37% of the normal serum total.
LDH 3 - Found in a variety of
organs and is 18% - 25% of the normal serum total.
LDH 4 - Found in a variety of
organs and is 3% - 8% of the normal serum total.
LDH 5 - Found in liver and
skeletal muscle and is 0% - 5% of the normal serum total.
Following
a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching a
peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a
comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than
CPK
is found primarily in heart and skeletal muscle as well as the brain.
Therefore, measurement of serum CPK levels is a good diagnostic for injury to
these tissues. The levels of CPK will rise within 6 hours of injury and peak by
around 18 hours. If the injury is not persistent the level of CK returns to
normal within 2-3 days. Like LDH, there are tissue-specific isozymes of CPK and
there designations are described below.
CPK3 (CPK-MM) is the
predominant isozyme in muscle and is 100% of the normal serum total.
CPK2 (CPK-MB) accounts for about
35% of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle
and is 0% of the normal serum total.
CPK1 (CPK-BB) is the
characteristic isozyme in brain and is in significant amounts in smooth muscle
and is 0% of the normal serum total.
Since
most of the released CPK after a myocardial infarction is CPK-MB, an increased
ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but
an increase of total CPK in itself may not. CPK-MB levels rise 3-6 hours after
a myocardial infarct and peak 12-24 hours later if no further damage occurs and
returns to normal 12-48 hours after the infarct.
CLINICAL
ENZYMOLOGY
http://www.youtube.com/watch?v=5hrU6_tic7s&feature=related
LACTATE DEHYDROGENASE (LDH)
(LD)
The total LDH
is generally tested by reaction of the serum sample with pyruvate and NADH2.
LDH will convert pyruvate to lactate, and in turn NADH is use up by the
reaction.
Normal
value of LDH in serum is 100-200 U/L. Values the upper range
are generally seen in children. Strenuous exercise will slightly increase the
value. LDH level is 100 times more inside the RBC than in plasma, and therefore
minor amount of hemolysis will result in a false-positive test. In myocardial
infarction, total LDH activity is increased, while H4 isoenzyme is
increased 5-10 times more.
Differential diagnosis: Increase
in total LDH level is seen in hemolytic anemias, hepatocellular damage,
muscular dystrophy, carcinomas, leukemias, and any condition which causes
necrosis of body cells. Since total LDH is increased in
many
conditions, the study of isozymes of LDH is of great importance.
Isoenzymes
of LDH
LDH enzyme is
a tetramer with four subunits. But the subunit may be either H (heart) or M
(muscle) polypeptide chains. These two are the products of two different genes.
Although both of them have the same molecular weight (32 kD), there are minor
amino acid variations. So five combinations of H and M chains are possible; H4,
H3M, H2M2, M3H and M4
varieties, forming five isoenzymes.
All these
five forms are seen in all persons. M4 form is seen in skeletal
muscles; it is not inhibited by pyruvate. But H4 form is seen in
heart and is inhibited by pyruvate. Normally LDH-2 (H3M1)
concentration in blood is greater than LDH-1 (H4); but this pattern is reversed
in myocardial infarction; this is called flipped pattern. The isoenzymes
are usual ly separated by cellulose acetate electrophoresis at pH 8.6.
They are then identified by adding the reactants finally producing a colour
reaction. . Lactate dehydrogenase isoenzymes (as percentage of total):
LDH1 14-26 %;
LDH2 29-39 %; LDH3 20-26 %;
LDH4 8-16%;
LDH5 6-16 %.
CREATINE
KINASE(CK)
http://www.youtube.com/watch?v=6r5Ddlcq26s
Creatine
→ Creatine phosphate
It was called
as creatine phosphokinase in old literature.
Normal
serum value for CK is 15-100 U/L for males and 10-80 U/L for
females.
CK
and Heart Attack CK value in serum is increased in myocardial
infarction. The CK level starts to rise within three hours of
infarction. Therefore, CK estimation is very useful to detect early cases,
where ECG changes may be ambiguous. The CK level is not increased in hemolysis
or in congestive cardiac failure; and therefore CK has an advantage over LDH.
CK
and Muscle Diseases he level of CK in serum is very much
elevated in muscular dystrophies (500 -1500 IU/L). The level is very high in the
early phases of the disease. In such patients a fall in CK level is indicative
of deteriorating condition, because by that time, all muscle mass is destroyed.
In female carriers of this X-linked disease (genotypically heterozygous), CK is
seen to be moderately raised. CK level is highly elevated in crush injury,
fracture and acute cerebrovascular accidents. Estimation of total CK is
employed in muscular dystrophies and MB iso-enzyme is estimated in myocardial
infarction.
Isoenzymes
of CK CK is a dimer; each subunit has a molecular weight of
40,000. The subunits are called B for brain and M for muscle. They are products
of loci in chromosomes 14 and 19 respectively. Therefore three isoenzymes are
seen in circulation. Normally CK2 is only 5% of the total activity.
Even doubling the value in CK2 (MB) iso-enzyme may not be detected,
if total value of CK alone is estimated. Hence the detection of MB-isoenzyme
is important in myocardial infarction. CK-MB < 6 % of total CK in
normal conditions.
The above
three isoenzymes are cytosolic. A fourth variety, called CK-mt is located in
mitochondria and constitutes about 15% of total CK activity. Its gene is
located in chromosome 15. CK1 may be complexed with immunoglobulin; and then
termed macroCK. CK1-lgG causes false-positive diagnosis of
myocardial infarction because it has an electrophoretic mobility close to CK2.
For quantitating MB isoenzyme, anti-MM antiserum is added to the patient's
serum. This will precipitate MM iso-enzyme. The supernatant serum is used for the
CK estimation. Here it is assumed that BB isoenzyme is negligible in quantity,
which is correct if there is no brain disease. CK iso-enzymes can also be
identified by electrophoresis.
ASPARTATE
AMINO TRANSFERASE (AST)
It is also called as serum glutamate-oxaloacetate
transaminase (SGOT). AST needs pyridoxal phosphate as co-enzyme. AST is
estimated by taking aspartate, α-ketoglutarate,
pyridoxal phosphate (vitamin B6) and patient' serum as the source of
AST. The oxaloacetate formed may be allowed to react with dinitrophenyl
hydrazine to produce a colour which is estimated colorimetrically at 520 nm.
Normal serum level of AST is
8-40 U/L or (0,1-0,45 mmol/(hour´L))
It is significantly elevated
in myocardial infarction. It if moderately elevated in liver diseases. However,
a marked increase in AST may be seen in primary hepatoma. AST has two
isoenzymes; cytoplasmic and mitochondrial. In mile degree of tissue injury,
cytoplasmic form is seen in serum. Mitochondrial type is seen in severe injury.
ALANINE
AMINO TRANSFERASE (ALT)
It is also
called as serum glutamate-pyruvate transaminase (SGPT). ALT needs pyridoxal
phosphate as coenzyme.
Normal serum level of ALT is
5-30 U/L or (0,1-0,68 mmol/(hour´L))
Very high values (100 to 1000 U/L)
are seen in acute hepatitis, either toxic or viral in origin. Both ALT and AST
are increased in liver diseases, but ALT >AST. Moderate increase (25 to 100
U/L) may be seen in chronic liver disease such as cirrhosis, and malignancy in
liver. A sudden fall in ALT level in cases of hepatitis is a very bad
prognostic sign. Ritis coefficient (AST/ALT) in normal conditions is 1,33±0,42.
http://www.youtube.com/watch?v=nXRWkorYFXc
ALKALINE PHOSPHATASE (ALP)
It
is a non- specific enzyme which hydrolyses aliphatic, aromatic or heterocyclic
compounds. The pH optimum for the enzyme reaction is between 9 and 10. It is
prodused by osteoblasts of bone, and localized in cell membranes.
Normal serum level of ALP is
40-125 U/L or 0,5-1,3 mmol/(hour´ L).
In children the upper level of
normal value may be more, becouse of the increased osteoblastic activity. Mild
increase is noticed during pregnancy, due to production of placental isoenzyme.
Moderate (2-3 times) increase
in ALP level is seen in hepatic diseases such as hepatitis, alcoholic hepatosis
or hepatocellular carcinoma. Very high levels of ALP (10-12 times of upper
limit) may be noticed in extrahepatic obstructions or cholestasis. ALP is
produced by epithelial cells of biliary canaliculi and obstruction of bile with
consequent irritation of epithelial cells leads to secretion of ALP into serum.
Drastically
high levels of ALP (10-25 times of upper limit) are also seen in bone diseases
where osteoblastic activity is enhanced such as Paget's disease, rickets,
osteomalacia, osteoblastoma, metastatic carcinoma of bone and
yperparathyroidism (Paget's disease or osteitis deformans was described in 1877
by Sir James Paget).
Isoenzymes
of Alkaline Phosphatase
1. α-1
ALP moves in α -1 position, it is synthesised by
epithelial cells of biliary canaliculi. It is about 10% of total activity and
is increased in obstructive jaundice and to some extent in metastatic carcinoma
of liver.
2. α
-2 heat labile ALP is stable at
3. α
-2 heat stable ALP will not be destroyed at
4. Pre-ß ALP is of bone
origin and elevated levels are seen in bone diseases. This is the most heat
labile (destroyed at
5. γ-ALP
is inhibited by phenylalanine and originates from intestinal cells. It is
increased in ulcerative colitis. About 10% of plasma ALP are of intestinal
variety.
6. The leucocyte alkaline
phosphatase (LAP) is significantly decreased in chronic myeloid leukemia. It is
increased in lymphomas.
ALP has
different isoforms. Although ALP is a monomer, depending on the number of
sialic acid residues, the charged groups differ. Such different forms are
detected in agar gel electrophoresis.
GAMMA
GLUTAMYL TRANSFERASE (GGT)
The old name was gamma glutamyl transpeptidase. It can
transfer γ-glutamyl residues to substrate. In
the body it is used in the synthesis of glutathione. GGT has 11 iso-enzymes. It
is seen in liver, kidney, pancreas, intestinal cells and prostate gland.
Normal
serum value of GGT is 6-45 U/L in male and 5-30 U/L in female. It is slightly
higher in normal males, due to the presence of prostate gland. This value is
moderately increased in infective hepatitis and prostate cancers. The GGT level
is highly elevated in alcoholism, obstructive jaundice and neoplasm's of liver.
GGT-2 is positive for 90% of hepatocellular carcinomas. It is not elevated in
cardiac or skeletal diseases.
GGT is a microsomal enzyme. Its activity is induced by
alcohol, phenobarbitone and rifampicin. GGT is clinically important because of
its sensitivity to detect alcohol abuse. GGT is increased in alcoholics even
when other liver function tests are within normal limits. GGT level is rapidly
decreased within a few days when the person stops to take alcohol. Increase in
GGT level is generally proportional to the amount of alcohol intake.
ACID
PHOSPHATASE (ACP)
It hydrolyses phosphoric acid ester at pH between 4
and 6. Methods for assay are the same as described for ALP; but the pH of the medium
is kept at 5 to 5.4.
Normal
serum value for ACP is 2.5-12 U/L or 0,025-0,12 mmol/(hour´ L).
ACP
is secreted by prostate cells, RBC, platelets and WBC. Isoenzymes of ACP are
described. Erythrocyte ACP gene is located in chromosome 2; osteoclast ACP gene
is on chromosome 19; lysosomal gene is on 11 and prostate ACP gene is on 13.
The prostate iso-enzyme is inactivated by tartaric acid. Cupric ions inhibit
erythrocyte ACP. Normal level of tartrate labile fraction of ACP is 1 U/L.
ACP
total value is increased in prostate cancer and highly elevated in bone
metastasis of prostate cancer. In these conditions, the tartrate labile
iso-enzyme is elevated. This assay is very helpful in follow up of treatment of
prostate cancers. ACP is therefore an
important tumour marker.
Since blood cells contain excess
quantity of ACP, must be taken to prevent hemolysis while taking blood from the
patient. Prostate massage may also increase to value. So blood may be collected
for ACP estimation before per rectal examination of patient. ACP is present in
high concentration in semen, a finding which is used in forensic medicine in
investigation of rape.
CHOLINESTERASE
(ChE)
Acetyl
cholinesterase or true ChE or Type 1 ChE can act mainly on acetyl choline.
It
is present in nerve endings and in RBCs. About 25 allelic forms are reported.
Normal
serum range is 2-12 U/ml. Newly formed RBC will contain good quantity of ChE
which is slowly reduced according to the age of the cell. Therefore, ChE level
in RBCs will be proportional to the reticulocyte count. Organophosphorus
insecticides (Parathione) irreversibly inhibit ChE in RBCs. Measurement of ChE
level in RBCs is useful to determine the amount of exposure in persons working
with these insecticides.
Pseudocholinesterase
or type II ChE is non-specific and can hydrolyse acyl esters. It is produced
mainly by liver cells. Normal serum level is 8-18 U/ml. Succinyl choline is a
widely used as muscle relaxant. It is a structural analogue of ACh, and so
competitively fix on post-synaptic receptors of ACh. Succinyl choline is
hydrolysed by the liver ChE within 2-4 minutes. But in certain persons the ChE
activity may be absent; this is a genetically transmitted condition. In such
individuals when succinyl choline is given during surgery, it may take hours to
get the drug metabolised. Very prolonged scoline apnoea may result in
'nightmare of anaesthetist'. The pseudocholinesterase level in serum is reduced
in viral hepatitis, cirrhosis, hepatocellular carcinoma, metastatic cancer of
liver and in malnutrition.
http://www.youtube.com/watch?v=-gIqZ8IxctE
GLUCOSE-6-PHOSPHATE
DEHYDROGENASE
GPD is a dimer with identical subunits. This is an important
enzyme in the hexose monophosphate shunt pathway of glucose. It is mainly used
for production of NADPH .
It
has a special role in the RBC metabolism. Due to the presence of oxygen,
hydrogen peroxide is continuously formed inside the RBC. Peroxide will destroy
biomembranes, and RBCs are lysed. Normal value of GPD in RBC is 125-250 U/1012
cells. Nearly 400 variants (isoforms) of GPD are described.
AMYLASE
This splits
starch to maltose.
It is activated by calcium, chloride and fluoride
ions. There are 18 phenotypes. It is produced by pancreas and salivary glands;
they are products of different genes located in chromosome 1.
http://www.youtube.com/watch?v=AEsQxzeAry8
Normal
serum value is 50-120 U/L, (12-32 g/(hour× L)).
The value is increased about 1000 times in acute
pancreatitis which is a life-threatening condition. The peak values are seen
between 5-12 hours after the onset of disease and returns to normal levels
within 2-4 days after the acute phase has subsided. Moderate increase in serum levels are seen in chronic pancreatitis,
mumps (parotitis), obstruction of pancreatic duct and in renal disease. In the
last condition, the enzyme is not excreted through urine properly and hence serum
value is raised. Normal urine value is 20-160 g/(hour× L) or (less than
375 U/L). It is increased in acute pancreatitis. It is increased on the
LIPASE
It will hydrolyse triglyceride to β-monoglyceride
and fatty acid. Molecular weight is 54,000.
The
gene is in chromosome 10. The enzyme is present in pancreatic secretion. Normal
serum range is 0.2-1.5 U/L. It is highly elevated in acute pancreatitis and
this persists for 7-14 days. Thus, lipase remains elevated longer than amylase.
Moreover, lipase is not increased in mumps. Therefore, lipase estimation has
advantage over amylase. It is moderately increased in carcinoma of pancreas,
biliary diseases and perforating peptic ulcers.
ALDOLASE (ALD)
It is a tetrameric enzyme with A
and B subunits; so there are 5 iso-enzymes. It is a glycolytic enzyme. Normal
range of serum is 1.5-7 U/L. It is drastically elevated in muscle damages such
as progressive muscular dystrophy, poliomyelitis, myasthenia gravis and multiple
sclerosis. It is a very sensitive early index in muscle wasting diseases.
ENOLASE
It is a glycolytic enzyme. Neuron-specific enolase
(NSE) is an iso-enzyme seen in neural tissues and Apudomas. NSE is a tumour
marker for cancers associated with neuro-endocrine origin, small cell lung
cancer, neuroblastoma, pheochromocytoma, medullary carcinoma of thyroid, etc.
It is measured by RIA or ELISA. Upper limit of NSE is 12 μg/ml.
Usage enzymes in medical practice
Enzymo-pathologies – disoders of enzymes action.
Enzymo-pathologies takes place actually at any disease (it’s so called
secondary enzymo-pathologies). But there also are more than one thousand
different forms of congenital molecular pathologies (primary
enzymo-pathologies). Mutations (genetic disorders) were found to be the causes
of these kind of enzymo-pathologies.
The examples of primary enzymo-pathologies:
1) Enzyme phenylalanine
4-monooxygenase is absent in about
The phenylalanine ,
along with seven amino acids (tyrosine, valine, tryptophan, lysine, isoleucine,
leucine and methionine) form a group called essential amino acids, which are
giving rise to other amino acids of which we are to obtain compounds and that
in the diet ingestion.
It is a hereditary disease with autosomal recessive
Both parents must have the defective gene.
The probability that the abnormal gene is passed on to the children is 75%:
o
25% chance of inheriting two defective genes and therefore suffer the disease.
o
50% have a defective copy that can pass to offspring and one normal copy: are
healthy carriers.
25% have no defective copy of the gene so it does not have the disease and can
transmit it to offspring.
The
probabilities are independent with each pregnancy.
2) The absence of homogentisic
acid 1,2-dioxygenase causes urinary excretion of phenylpyruvic and
homogentisic acids. The urinary of people genetically defective in homogentisic
acid 1,2-dioxygenase contains homogentisic acid, which when made alkaline and
exposed to oxygen, turns dark because it is oxidized and polimerized to a black
melanin pigment.
This condition is known as alkaptonuria.
Patients with this condition have abnormal pigmentation of the connective
tissue.
3) D-galactose is converted into
D-glucose in the liver by special reactions which have attracted much attention
because they are subject to genetic defects in man, resulting in different
forms of the hereditary disease galactosemia. The deffect or absence of
enzyme galactose 1-phosphate uridylyltransferase causes the increase of
D-galactose in the blood. Galactose 1-phosphate uridylyltransferase is
present in normal fetal liver but is lacking in infants with galactosemia.
Galactosemic infants suffer from cataract of the lens of the eye as well as
mental disorders. This condition can be successfully treated by withholding
milk and other sources of galactose from the diet during infancy and childhood.
The physical symptoms of this disease varies with
what kind of galactosemia you have. There are three types: Classic Galactosemia
(type I), Galactosemia type II, and Galactosemia type III.
Symptoms of
Classic Galactosemia:
Cataracts
When babies are born and they
find out that they have Galactosemia, they get the kind of diet they need and
live a normal life but if they don't it could be life -threatening. They could
have a lack of energy, failure to eat and grow. Also their skin will become
yellow, their eyes will be white , and liver damage. That is if they have the
classic Galactosemia or type I. Galactosemia can not be treated but there is
steps you can take to help you with galacatosemia. The most simple treatment is
to watch what you eat.
Enzymo-therapy. In digestive tract diseases
(deficit of digestive enzymes) pepsin and HCI, enzymes of pancreas (pancreatin,
oraza, panzinorm) are recomended to use.
Proteolytic enzymes trypsin and
chemotrypsin are used for processing of wounds in burns, ulcers for the a proteolysis and deleting of necrotic tissues.
Trypsin, ribonuclease,
DNA-ase are used for the splitting and deleting of fibrin from the pleural
cavity, for the dilution and deleting of sputum from the respiratory ways in acute
and chronic respiratory diseases, for the thrombophlebitis treatment.
Fibrinolysin,
streptokinase are used for the splitting of thrombus.
Hyaluronidase (lidase) is used for
the acceleration of different medicines penetration into the biological tissues
as well as for the resorption of scars and
hematoma.
Cytochrome C is used in
the intoxication of CO, H2S and other substances oppressing the
tissue respiration.
Glucoso-oxidase is used
for the lavage of wounds and burns as antiseptic.
Thrombin is used for
preventing and stop of bleeding.
The
using of coenzymes. ATP is used in heart diseases, muscle
dystrophia.
TPP
(thiamin pyrophosphate) is used in heart diseases, and nervous system
pathology.
FMN
(flavinmononucleotide) is used for treatment of skin diseases, keratitis,
conjunctivitis.
NAD
and NADP are used for improvement of oxidative-reduction processes in organism.
The coenzyme folic acid (left) and
the anti-cancer drug methotrexate (right) are very similar in structure. As a
result, methotrexate is a competitive inhibitor of many enzymes that use
folates.
In 1897 Eduard Buchner began to
study the ability of yeast extracts to ferment sugar despite the absence of
living yeast cells. In a series of experiments at the
Having shown that enzymes could function outside a
living cell, the next step was to determine their biochemical nature. Many
early workers noted that enzymatic activity was associated with proteins, but
several scientists (such as Nobel laureate Richard Willstätter) argued
that proteins were merely carriers for the true enzymes and that proteins per
se were incapable of catalysis. However, in 1926, James B. Sumner showed that
the enzyme urease was a pure protein and crystallized it; Sumner did likewise
for the enzyme catalase in 1937. The conclusion that pure proteins can be
enzymes was definitively proved by Northrop and Stanley, who worked on the
digestive enzymes pepsin (1930), trypsin and chymotrypsin. These three
scientists were awarded the 1946 Nobel Prize in Chemistry.
This discovery that enzymes could be crystallized
eventually allowed their structures to be solved by x-ray crystallography. This
was first done for lysozyme, an enzyme found in tears, saliva and egg whites
that digests the coating of some bacteria; the structure was solved by a group
led by David Chilton Phillips and published in 1965. This high-resolution
structure of lysozyme marked the beginning of the field of structural biology
and the effort to understand how enzymes work at an atomic level of detail.
Today, the findings of biochemistry are used in many areas, from genetics to molecular biology and from agriculture to medicine.
Characteristics of
Enzymes
Enzymes are
proteins that catalyze (i.e. accelerate) chemical reactions. In these
reactions, the molecules at the beginning of the process are called substrates,
and the enzyme converts them into different molecules, the products. Almost all
processes in a biological cell need enzymes in order to occur at significant
rates. Since enzymes are extremely selective for their substrates and speed up
only a few reactions from among many
possibilities, the set of enzymes made in a cell determines which metabolic
pathways occur in that cell.
Like all catalysts, enzymes work by lowering the
activation energy (ΔG‡)
for a reaction, thus dramatically accelerating the rate of the reaction. Most
enzyme reaction rates are millions of times faster than those of comparable
uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the
reactions they catalyze, nor do they alter the equilibrium of these reactions.
However, enzymes do differ from most other catalysts by being much more
specific. Enzymes are known to catalyze about 4,000 biochemical reactions. Not
all biochemical catalysts are proteins, since some RNA molecules called
ribozymes also catalyze reactions.
http://www.youtube.com/watch?v=AFbPHlhI13g&feature=related
http://www.youtube.com/watch?v=AEsQxzeAry8&feature=related
http://www.youtube.com/watch?v=KED6BHVM97s&feature=related
Enzymes
are protein molecules that are tailored to recognize and bind specific
reactants and speed their conversion into products. These proteins are
responsible for increasing the rates of all of the many thousand of reaction
taking place inside cells.
All enzymatic proteins have several characteristics in common
table 1.
Table 1: Characteristics of enzymes proteins
1. |
Enzymes combine briefly with
reactants during an enzyme-catalyzed reaction. |
2. |
Enzymes are released unchanged
after catalyzing the conversion of reactants to Product |
3. |
Enzymes are specific in their
activity; each enzyme catalyzes the reaction of a single type of molecules or
a group of closely related molecules. |
4. |
Enzymes are saturated by high
substrate concentrations. |
5. |
Many enzymes contain nonproteins groups
called cofactors, which contribute to their activity. Inorganic cofactors are
all metallic ions. Organic cofactors, called coenzymes, are complex groups
derived from vitamins. |
6. |
Many enzymes are pH and temperature
sensitive |
The rate of combination
and release, known as the turnover number, lies near 1000 per second for most
enzymes. Some enzymes have turnover numbers as small as 100 per second or as
large as 10 million per second. As a result of enzyme turnover, a relatively small
number of enzyme molecules can catalyze a large number of reactant molecules.
The part
of an enzyme that combines with substrate molecule is the active site. In most
enzymes the active site is located in a cavity or pocket on the enzyme surface,
frequently within a cleft marking the boundary between two or more major
domains. Within the cleft or pocket, amino acid side groups are situated to fit
and bind parts of substrate molecules that are critical to the reaction
catalyzed by the enzyme. The active site also separates substrate molecules
from the surrounding solutions and place them in environments with unique
characteristics, including partial or complete exclusion of water.
Enzymes
as Biological Catalysts
In cells and organisms most reactions are
catalyzed by enzymes, which are regenerated during the course of a reaction.
These biological catalysts are physiologically important because they speed up
the rates of reactions that would otherwise be too slow to support life.
Enzymes increase reaction rates--- sometimes by as much as one millionfold, but
more typically by about one thousand fold. Catalysts speed up the forward and
reverse reactions proportionately so that, although the magnitude of the rate
constants of the forward and reverse reactions is are increased, the ratio of
the rate constants remains the same in the presence or absence of enzyme. Since
the equilibrium constant is equal to a ratio of rate constants, it is apparent
that enzymes and other catalysts have no effect on the equilibrium constant of
the reactions they catalyze.
Enzyme activity can be affected by
other molecules. Inhibitors are molecules that decrease enzyme activity;
activators are molecules that increase activity. Many drugs and poisons are
enzyme inhibitors. Activity is also affected by temperature, pH, and the
concentration of substrate. Some enzymes are used commercially, for example, in
the synthesis of antibiotics. In addition, some household products use enzymes
to speed up biochemical reactions (e.g., enzymes in biological washing powders
break down protein or fat stains on clothes; enzymes in meat tenderizers break
down proteins, making the meat easier to chew).
http://www.youtube.com/watch?v=cXLpxe6sPwI&feature=related
Most enzymes are much larger than the
substrates they act on, and only a very small portion of the enzyme (around 3–4
amino acids) is directly involved in catalysis.
http://www.youtube.com/watch?v=0pJOFze055Y
The region that contains these
catalytic residues, binds the substrate, and then carries out the reaction is
known as the active site. Enzymes can also contain sites that bind cofactors,
which are needed for catalysis. Some enzymes also have binding sites for small
molecules, which are often direct or indirect products or substrates of the
reaction catalyzed.
Ribbon-diagram showing carbonic anhydrase II. The grey
sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO.The
activities of enzymes are determined by their three-dimensional structure.
This binding can serve to increase or decrease the enzyme's activity,
providing a means for feedback regulation. Enzymes are catalysts. Most are proteins. (A few ribonucleoprotein enzymes
have been discovered and, for some of these, the catalytic activity is in the
RNA part rather than the protein part. Link to discussion of these ribozymes.)
Enzymes bind
temporarily to one or more of the reactants
of the reaction they catalyze. In doing so, they lower the amount of activation
energy needed and thus speed up the reaction.
Examples:
· Catalase. It catalyzes the decomposition of hydrogen peroxide into water and
oxygen.
2H2O2
-> 2H2O + O2
One
molecule of catalase can break 40 million molecules of hydrogen peroxide each
second.
· Carbonic anhydrase. It is found in red blood cells where it
catalyzes the reaction
CO2 + H2O
<-> H2CO3
It enables
red blood cells to transport carbon dioxide from the tissues to the lungs. One
molecule of carbonic anhydrase can process one million molecules of CO2
each second.
Like all proteins, enzymes are made
as long, linear chains of amino acids that fold to produce a three-dimensional
product. Each unique amino acid sequence produces a unique structure, which has
unique properties. Individual protein chains may sometimes group together to
form a protein complex. Most enzymes can be denatured—that is, unfolded and
inactivated—by heating, which destroys the three-dimensional structure of the
protein. Depending on the enzyme, denaturation may be reversible or
irreversible.
Specificity
Enzymes are usually very specific as
to which reactions they catalyze and the substrates that are involved in these
reactions. Complementary shape, charge and hydrophilic/hydrophobic
characteristics of enzymes and substrates are responsible for this specificity.
Enzymes can also show impressive levels of stereospecificity, regioselectivity
and chemoselectivity.
Some of the enzymes showing the
highest specificity and accuracy are involved in the copying and expression of
the genome. These enzymes have "proof-reading" mechanisms. Here, an
enzyme such as DNA polymerase catalyses a reaction in a first step and then
checks that the product is correct in a second step. This two-step process
results in average error rates of less than 1 error in 100 million reactions in
high-fidelity mammalian polymerases. Similar proofreading mechanisms are also
found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.
Some enzymes that produce secondary metabolites are
described as promiscuous, as they can act on a relatively broad range of
different substrates. It has been suggested that this broad substrate
specificity is important for the evolution of new biosynthetic pathways.
"Lock and key" model
Enzymes are very specific, and it was
suggested by Emil Fischer in 1894 that this was because both the enzyme and the
substrate possess specific complementary geometric shapes that fit exactly into
one another. This is often referred to as "the lock and key" model.
However, while this model explains enzyme specificity, it fails to explain the
stabilization of the transition state that enzymes achieve.
Induced fit model
Diagrams to
show the induced fit hypothesis of enzyme action.In 1958 Daniel Koshland
suggested a modification to the lock and key model: since enzymes are rather
flexible structures, the active site can be reshaped by interactions with the
substrate as the substrate interacts with the enzyme. As a result, the
substrate does not simply bind to a rigid active site, the amino acid side
chains which make up the active site are molded into the precise positions that
enable the enzyme to perform its catalytic function. In some cases, such as
glycosidases, the substrate molecule also changes shape slightly as it enters
the active site.
Providing an alternative pathway
(e.g. temporarily reacting with the substrate to form an intermediate ES
Complex which would be impossible in the absence of the enzyme).
Reducing the reaction entropy change
by bringing substrates together in the correct orientation to react.
Considering ΔH‡
alone overlooks this effect.
Dynamics and
function
Recent investigations
have provided new insights into the connection between internal dynamics of
enzymes and their mechanism of catalysis. An enzyme's internal dynamics are described as the
movement of internal parts (e.g. amino acids, a group of amino acids, a loop
region, an alpha helix, neighboring beta-sheets or even entire domain) of these
biomolecules, which can occur at various time-scales ranging from femtoseconds
to seconds. Networks of protein residues throughout an enzyme's structure can
contribute to catalysis through dynamic motions. Protein motions are vital to many enzymes, but
whether small and fast vibrations or larger and slower conformational movements
are more important depends on the type of reaction involved. These new insights also have implications in understanding allosteric
effects, producing designer enzymes and developing new drugs.
Cofactors and coenzymes
Role of Coenzymes
The functional role of coenzymes is to act as
transporters of chemical groups from one reactant to another. The chemical
groups carried can be as simple as the hydride ion (H+ + 2e-) carried by NAD or
the mole of hydrogen carried by FAD; or they can be even more complex than the
amine (-NH2) carried by pyridoxal phosphate.
Since coenzymes are chemically changed as a consequence of enzyme
action, it is often useful to consider coenzymes to be a special class of
substrates, or second substrates, which are common to many different
holoenzymes. In all cases, the coenzymes donate the carried chemical grouping
to an acceptor molecule and are thus regenerated to their original form. This
regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as
a chemical catalyst, since (unlike the usual substrates, which are used up
during the course of a reaction) coenzymes are generally regenerated.
Enzyme Relative to Substrate Type
Enzymes also are generally specific for a
particular steric configuration (optical isomer) of a substrate. Enzymes that
attack D sugars will not attack the corresponding L isomer. Enzymes that act on
L amino acids will not employ the corresponding D optical isomer as a
substrate. The enzymes known as racemases provide a striking exception to these
generalities; in fact, the role of racemases is to convert D isomers to L
isomers and vice versa. Thus racemases attack both D and L forms of their
substrate.
As enzymes have a more or less broad range of substrate specificity, it
follows that a given substrate may be acted on by a number of different
enzymes, each of which uses the same substrate(s) and produces the same
product(s). The individual members of a set of enzymes sharing such
characteristics are known as isozymes. These are the products of genes that
vary only slightly; often, various isozymes of a group are expressed in different
tissues of the body. The best studied set of isozymes is the lactate
dehydrogenase (LDH) system. LDH is a tetrameric enzyme composed of all possible
arrangements of two different protein subunits; the subunits are known as H
(for heart) and M (for skeletal muscle). These subunits combine in various
combinations leading to 5 distinct isozymes. The all H isozyme is
characteristic of that from heart tissue, and the all M isozyme is typically
found in skeletal muscle and liver. These isozymes all catalyze the same
chemical reaction, but they exhibit differing degrees of efficiency. The
detection of specific LDH isozymes in the blood is highly diagnostic of tissue
damage such as occurs during cardiac infarct.
Many enzymes require the presence of
an additional, nonprotein, cofactor.
· Some of these are metal ions such as Zn2+ (the cofactor for
carbonic anhydrase), Cu2+, Mn2+, K+, and Na+.
· Some cofactors are small organic molecules called coenzymes. The B vitamins
o
thiamine (B1)
o
riboflavin (B2) and
are
precursors of coenzymes.
Coenzymes
may be covalently bound to the protein part (called the apoenzyme) of
enzymes as a prosthetic group. Others bind more loosely and, in fact,
may bind only transiently to the enzyme as it performs its catalytic act.
Lysozyme:
a model of enzyme action
A number of lysozymes are found in nature; in human
tears and egg white, for examples. The enzyme is antibacterial because it
degrades the polysaccharide that is found in the cell walls of many bacteria.
It does this by catalyzing the insertion of a water molecule at the position
indicated by the red arrow. This hydrolysis breaks the chain at that point.
The bacterial polysaccharide consists of long chains
of alternating amino sugars:
· N-acetylglucosamine (NAG)
· N-acetylmuramic acid (NAM)
These hexose units resemble glucose except for the presence of the side
chains containing amino groups.
Lysozyme is a globular
protein with a deep cleft across part of its surface. Six hexoses of the
substrate fit into this cleft.
· With so many oxygen atoms in sugars, as many as 14 hydrogen
bonds form between the six amino sugars
and certain amino acid R
groups such as Arg-114, Asn-37,
Asn-44, Trp-62, Trp-63, and Asp-101.
· Some hydrogen bonds also form with the C=O groups of several peptide
bonds.
· In addition, hydrophobic interactions may help hold the substrate in
position.
As for
lysozyme itself, binding of the substrate induces a small (~0.75Å)
movement of certain amino acid residues so the cleft closes slightly over its
substrate. So the "lock" as well as the "key" changes shape
as the two are brought together. (This is sometimes called "induced
fit".)
The amino
acid residues in the vicinity of rings 4 and 5 provide a plausible mechanism
for completing the catalytic act. Residue 35, glutamic acid (Glu-35), is
about 3Å from the -O- bridge that is to be broken. The free carboxyl group
of glutamic acid is a hydrogen ion donor and available to transfer H+
to the oxygen atom. This would break the already-strained bond between the
oxygen atom and the carbon atom of ring 4.
Now having lost an
electron, the carbon atom acquires a positive charge. Ionized carbon is
normally very unstable, but the attraction of the negatively-charged carboxyl
ion of Asp-52 could stabilize it long enough for an -OH ion (from a
spontaneously dissociated water molecule) to unite with the carbon. Even at pH
7, water spontaneously dissociates to produce H+ and OH-
ions. The hydrogen ion (H+) left over can replace that lost by Glu-35.
In either case, the chain
is broken, the two fragments separate from the enzyme, and the enzyme is free
to attach to a new location on the bacterial cell wall and continue its work of
digesting it.
Cofactors
Some enzymes do not need any
additional components to show full activity. However, others require
non-protein molecules to be bound for activity. Cofactors can be either inorganic
(e.g., metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin
and heme). Organic cofactors (coenzymes) are usually prosthetic groups, which
are tightly bound to the enzymes that they assist. These tightly-bound
cofactors are distinguished from other coenzymes, such as NADH, since they are
not released from the active site during the reaction.
An example of an enzyme that contains
a cofactor is carbonic anhydrase, and is shown in the ribbon diagram above with
a zinc cofactor bound in its active site. These tightly-bound molecules are
usually found in the active site and are involved in catalysis. For example,
flavin and heme cofactors are often involved in redox reactions.
Enzymes that require a cofactor but do not have one
bound are called apoenzymes. An apoenzyme together with its cofactor(s) is
called a holoenzyme (i.e., the active form). Most cofactors are not covalently
attached to an enzyme, but are very tightly bound. However, organic prosthetic
groups can be covalently bound (e.g., thiamine pyrophosphate in the enzyme
pyruvate dehydrogenase).
Coenzymes
Space-filling model of the coenzyme
NADH Coenzymes are small molecules that transport chemical groups from one enzyme
to another. Some of these chemicals such as riboflavin, thiamine and folic acid
are vitamins, this is when these compounds cannot be made in the body and must
be acquired from the diet. The chemical groups carried include the hydride ion
(H-) carried by NAD or NADP+, the acetyl group carried by coenzyme A, formyl,
methenyl or methyl groups carried by folic acid and the methyl group carried by
S-adenosylmethionine.
Since coenzymes are chemically changed as a
consequence of enzyme action, it is useful to consider coenzymes to be a
special class of substrates, or second substrates, which are common to many
different enzymes. For example, about 700 enzymes are known to use the coenzyme
NADH.
Coenzymes are usually regenerated and their
concentrations maintained at a steady level inside the cell: for example, NADPH
is regenerated through the pentose phosphate pathway and S-adenosylmethionine
by methionine adenosyltransferase.
Factors
Affecting Enzyme Action
The activity of enzymes is strongly
affected by changes in pH and temperature. Each enzyme works best
at a certain pH (left graph) and temperature (right graph), its activity
decreasing at values above and below that point. This
is not surprising considering the importance of
· tertiary
structure (i.e. shape) in enzyme function and
· noncovalent forces, e.g., ionic interactions and hydrogen bonds, in
determining that shape.
Examples:
· the protease pepsin works best as a pH of 1-2 (found in the
stomach) while
· the protease trypsin is inactive at such a low pH but very active
at a pH of 8 (found in the small intestine as the bicarbonate of the pancreatic
fluid neutralizes the arriving stomach contents).
Changes in pH alter the
state of ionization of charged amino acids (e.g., Asp, Lys) that may play a
crucial role in substrate binding and/or the catalytic action itself. Without
the unionized -COOH group of Glu-35 and the ionized -COO- of Asp-52,
the catalytic action of lysozyme would cease.
Hydrogen bonds are easily
disrupted by increasing temperature. This, in turn, may disrupt the shape of
the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve (red
arrow in right-hand graph above) reflects the general effect of increasing
temperature on the rate of chemical reactions (graph at left). The
descending portion of the curve above (blue arrow) reflects the loss of
catalytic activity as the enzyme molecules become denatured at high
temperatures.
Several mechanisms work to make
enzyme activity within the cell efficient and well-coordinated.
Many enzymes are inserted into cell
membranes, for examples,
· the plasma membrane
· the membranes of mitochondria and chloroplasts
· the endoplasmic reticulum
· the nuclear envelope
These are locked into spatial
relationships that enable them to interact efficiently.
Enzymes, such as proteases, that can
attack the cell itself are inhibited while within the cell that synthesizes
them. For example, pepsin is synthesized within the chief
cells (in gastric glands) as an inactive
precursor, pepsinogen. Only when exposed to the low pH outside the cell
is the inhibiting portion of the molecule removed and active pepsin produced.
If the product of a series of enzymatic reactions,
e.g., an amino acid, begins to accumulate within the cell, it may specifically
inhibit the action of the first enzyme involved in its synthesis (red bar).
Thus further production of the enzyme is halted.
The accumulation of a substance
within a cell may specifically activate (blue arrow) an enzyme that sets in
motion a sequence of reactions for which that substance is the initial
substrate. This reduces the concentration of the initial substrate.
In the
case if feedback inhibition and precursor activation, the activity of the enzyme
is being regulated by a molecule which is not its substrate. In these
cases, the regulator molecule binds to the enzyme at a different site than the
one to which the substrate binds. When the regulator binds to its site, it
alters the shape of the enzyme so that its activity is changed. This is called
an allosteric effect.
· In feedback inhibition, the allosteric effect lowers the affinity of the
enzyme for its substrate.
·
In precursor activation, the regulator
molecule increases the affinity of the enzyme in the series for its
substrate.If, for example, ample quantities of an amino acid are already
available to the cell from its extracellular fluid, synthesis of the enzymes
that would enable the cell to produce that amino acid for itself is shut down.
Conversely, if a new
substrate is made available to the cell, it may induce the synthesis of the
enzymes needed to cope with it. Yeast cells, for example, do not ordinarily
metabolize lactose and no lactase can be detected in them. However, if
grown in a medium containing lactose, they soon begin synthesizing lactase - by
transcribing and translating the necessary gene(s) - and so can begin to
metabolize the sugar.
Е. coli also has a
mechanism which regulates enzyme synthesis by controlling translation of
a needed messenger RNA..
Thermodynamics
Main aticles: Activation energy,
Thermodynamic equilibrium, and Chemical equilibrium.
Diagram of a
catalytic reaction, showing the energy niveau at each stage of the reaction.
The substrates usually need a large amount of energy to reach the transition
state, which then decays into the end product. The enzyme stabilizes the
transition state, reducing the energy needed to form this species and thus
reducing the energy required to form products.As all catalysts, enzymes do not
alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same
direction as it would without the enzyme, just more quickly. However, in the
absence of the enzyme, other possible uncatalyzed, "spontaneous"
reactions might lead to different products, because in those conditions this
different product is formed faster.
Furthermore, enzymes can couple two or more reactions,
so that a thermodynamically favorable reaction can be used to "drive"
a thermodynamically unfavorable one. For example, the hydrolysis of ATP is
often used to drive other chemical reactions.
Enzymes catalyze the forward and
backward reactions equally. They do not alter the equilibrium itself, but only
the speed at which it is reached. For example, carbonic anhydrase catalyzes its
reaction in either direction depending on the concentration of its reactants.
(in tissues; high CO2 concentration)
(in lungs; low CO2 concentration)
Nevertheless, if the equilibrium is greatly displaced
in one direction, that is, in a very exergonic reaction, the reaction is
effectively irreversible. Under these conditions the enzyme will, in fact, only
catalyze the reaction in the thermodynamically allowed direction.
Kinetics
Mechanism for a single substrate
enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a
product (P).Enzyme kinetics is the investigation of how enzymes bind substrates
and turn them into products. The rate data used in kinetic analyses are
obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed
a quantitative theory of enzyme kinetics, which is referred to as
Michaelis-Menten kinetics. Their work was further developed by G. E. Briggs and
J. B. S. Haldane, who derived kinetic equations that are still widely used
today.
Michaelis-Menton Kinetics
In typical enzyme-catalyzed reactions, reactant
and product concentrations are usually hundreds or thousands of times greater
than the enzyme concentration. Consequently, each enzyme molecule catalyzes the
conversion to product of many reactant molecules. In biochemical reactions,
reactants are commonly known as substrates. The catalytic event that converts
substrate to product involves the formation of a transition state, and it
occurs most easily at a specific binding site on the enzyme. This site, called
the catalytic site of the enzyme, has been evolutionarily structured to provide
specific, high-affinity binding of substrate(s) and to provide an environment
that favors the catalytic events. The complex that forms, when substrate(s) and
enzyme combine, is called the enzyme substrate (ES) complex. Reaction products
arise when the ES complex breaks down releasing free enzyme.
Between the binding of substrate to enzyme, and the reappearance of free
enzyme and product, a series of complex events must take place. At a minimum an
ES complex must be formed; this complex must pass to the transition state
(ES*); and the transition state complex must advance to an enzyme product
complex (EP). The latter is finally competent to dissociate to product and free
enzyme. The series of events can be shown thus:
E + S <---> ES <---> ES* <---> EP
<---> E + P
The kinetics of simple
reactions like that above were first characterized by biochemists Michaelis and
Menten. The concepts underlying their analysis of enzyme kinetics continue to
provide the cornerstone for understanding metabolism today, and for the development
and clinical use of drugs aimed at selectively altering rate constants and
interfering with the progress of disease states. The Michaelis-Menten equation
is a quantitative description of the relationship among the rate of an enzyme-
catalyzed reaction [v1], the concentration of substrate [S] and two constants,
Vmax and Km (which are set by the particular equation). The symbols used in the
Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction
rate (Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).
The
Michaelis-Menten equation can be used to demonstrate that at the substrate
concentration that produces exactly half of the maximum reaction rate, i.e.,1/2
Vmax, the substrate concentration is numerically equal to Km. This fact
provides a simple yet powerful bioanalytical tool that has been used to
characterize both normal and altered enzymes, such as those that produce the
symptoms of genetic diseases. Rearranging the Michaelis-Menton equation leads
to:
From this equation it should be apparent that when the substrate
concentration is half that required to support the maximum rate of reaction,
the observed rate, v1, will, be equal to Vmax divided by 2; in other words, v1
= [Vmax/2]. At this substrate concentration Vmax/v1 will be exactly equal to 2,
with the result that:
[S](1) = Km
The latter is an algebraic statement of the fact
that, for enzymes of the Michaelis-Menten type, when the observed reaction rate
is half of the maximum possible reaction rate, the substrate concentration is
numerically equal to the Michaelis-Menten constant. In this derivation, the
units of Km are those used to specify the concentration of S, usually Molarity.
The Michaelis-Menten equation has the same form
as the equation for a rectangular hyperbola; graphical analysis of reaction
rate (v) versus substrate concentration [S] produces a hyperbolic rate plot.
Plot of substrate concentration
versus reaction velocity
The key features of the plot are marked
by points A, B and C. At high substrate concentrations the rate represented by
point C the rate of the reaction is almost equal to Vmax, and the difference in
rate at nearby concentrations of substrate is almost negligible. If the
Michaelis-Menten plot is extrapolated to infinitely high substrate
concentrations, the extrapolated rate is equal to Vmax. When the reaction rate
becomes independent of substrate concentration, or nearly so, the rate is said
to be zero order. (Note that the reaction is zero order only with respect to
this substrate. If the reaction has two substrates, it may or may not be zero
order with respect to the second substrate). The very small differences in
reaction velocity at substrate concentrations around point C (near Vmax) reflect
the fact that at these concentrations almost all of the enzyme molecules are
bound to substrate and the rate is virtually independent of substrate, hence
zero order. At lower substrate concentrations, such as at points A and B, the
lower reaction velocities indicate that at any moment only a portion of the
enzyme molecules are bound to the substrate. In fact, at the substrate
concentration denoted by point B, exactly half the enzyme molecules are in an
ES complex at any instant and the rate is exactly one half of Vmax. At
substrate concentrations near point A the rate appears to be directly
proportional to substrate concentration, and the reaction rate is said to be
first order.
Inhibition of Enzyme Catalyzed Reactions
To avoid dealing with curvilinear plots of enzyme
catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of
enzyme kinetics based on the following rearrangement of the Michaelis-Menten
equation:
[1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]
Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax
and an intercept on the ordinate at 1/Vmax.
A Lineweaver-Burk Plot
An alternative linear transformation of the
Michaelis-Menten equation is the Eadie-Hofstee transformation:
v/[S] = -v [1/Km] + [Vmax/Km]
and when v/[S] is plotted on the y-axis versus v on the x-axis, the
result is a linear plot with a slope of -1/Km and the value Vmax/Km as the
intercept on the y-axis and Vmax as the intercept on the x-axis.
Both the Lineweaver-Burk and
Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the
analysis of enzyme inhibition. Since most clinical drug therapy is based on
inhibiting the activity of enzymes, analysis of enzyme reactions using the
tools described above has been fundamental to the modern design of
pharmaceuticals. Well- known examples of such therapy include the use of
methotrexate in cancer chemotherapy to semi-selectively inhibit DNA synthesis
of malignant cells, the use of aspirin to inhibit the synthesis of
prostaglandins which are at least partly responsible for the aches and pains of
arthritis, and the use of sulfa drugs to inhibit the folic acid synthesis that
is essential for the metabolism and growth of disease-causing bacteria. In
addition, many poisons, such as cyanide, carbon monoxide and polychlorinated
biphenols (PCBs). produce their life- threatening effects by means of enzyme
inhibition.
The major contribution of Michaelis and Menten was to
think of enzyme reactions in two stages. In the first, the substrate binds
reversibly to the enzyme, forming the enzyme-substrate complex. This is
sometimes called the Michaelis-Menten complex in their honor. The enzyme then
catalyzes the chemical step in the reaction and releases the product.
Saturation curve
for an enzyme reaction showing the relation between the substrate concentration
(S) and rate (v).Enzymes can catalyze up to several million reactions per
second. For example, the reaction catalyzed by orotidine 5'-phosphate
decarboxylase will consume half of its substrate in 78 million years if no
enzyme is present. However, when the decarboxylase is added, the same process
takes just 25 milliseconds. Enzyme rates depend on solution conditions and
substrate concentration. Conditions that denature the protein abolish enzyme
activity, such as high temperatures, extremes of pH or high salt
concentrations, while raising substrate concentration tends to increase
activity. To find the maximum speed of an enzymatic reaction, the substrate
concentration is increased until a constant rate of product formation is seen.
This is shown in the saturation curve, shown on the right. Saturation happens because, as substrate concentration increases, more
and more of the free enzyme is converted into the substrate-bound ES form. At
the maximum velocity (Vmax) of the enzyme, all enzyme active sites are
saturated with substrate, and the amount of ES complex is the same as the total
amount of enzyme.
However, Vmax is only one kinetic constant of enzymes.
The amount of substrate needed to achieve a given rate of reaction is also
important. This is given by the Michaelis-Menten constant (Km), which is the
substrate concentration required for an enzyme to reach one-half its maximum
velocity. Each enzyme has a characteristic Km for a given substrate, and this
can show how tight the binding of the substrate is to the enzyme. Another
useful constant is kcat, which is the number of substrate molecules handled by
one active site per second.
The defficiency of an enzyme can be expressed in terms
of kcat/Km. This is also called the specificity constant and incorporates the
rate constants for all steps in the reaction. Because the specificity constant
reflects both affinity and catalytic ability, it is useful for comparing
different enzymes against each other, or the same enzyme with different
substrates. The theoretical maximum for the specificity constant is called the
diffusion limit and is about 108 to 109 (M-1 s-1). At this point every
collision of the enzyme with its substrate will result in catalysis, and the
rate of product formation is not limited by the reaction rate but by the
diffusion rate. Enzymes with this property are called catalytically perfect or
kinetically perfect. Example of such enzymes are triose-phosphate isomerase,
carbonic anhydrase, acetylcholinesterase, catalase, fumarase,
ß-lactamase, and superoxide dismutase.
Some enzymes operate with kinetics which are faster
than diffusion rates, which would seem to be impossible. Several mechanisms
have been invoked to explain this phenomenon. Some proteins are believed to
accelerate catalysis by drawing their substrate in and pre-orienting them by
using dipolar electric fields. Other models invoke a quantum-mechanical
tunneling explanation, whereby a proton or an electron can tunnel through
activation barriers, although for proton tunneling this model remains somewhat
controversial. Quantum tunneling for protons has been observed in tryptamine.
This suggests that enzyme catalysis may be more accurately characterized as
"through the barrier" rather than the traditional model, which
requires substrates to go "over" a lowered energy barrier.
Enzyme inhibitors fall into two broad classes: those causing
irreversible inactivation of enzymes and those whose inhibitory effects can be
reversed. Inhibitors of the first class usually cause an inactivating, covalent
modification of enzyme structure. Cyanide is a classic example of an
irreversible enzyme inhibitor: by covalently binding mitochondrial cytochrome
oxidase, it inhibits all the reactions associated with electron transport. The
kinetic effect of irreversible inhibitors is to decrease the concentration of
active enzyme, thus decreasing the maximum possible concentration of ES
complex. Since the limiting enzyme reaction rate is often k2[ES], it is clear
that under these circumstances the reduction of enzyme concentration will lead
to decreased reaction rates. Note that when enzymes in cells are only partially
inhibited by irreversible inhibitors, the remaining unmodified enzyme molecules
are not distinguishable from those in untreated cells; in particular, they have
the same turnover number and the same Km. Turnover number, related to Vmax, is
defined as the maximum number of moles of substrate that can be converted to
product per mole of catalytic site per second. Irreversible inhibitors are
usually considered to be poisons and are generally unsuitable for therapeutic
purposes.
Reversible inhibitors can be divided
into two main categories; with a third
category, uncompetitive inhibitors, rarely encountered.
Inhibitor Type |
Binding Site on Enzyme |
Kinetic effect |
Competitive Inhibitor |
Specifically
at the catalytic site, where it competes with substrate for binding in a dynamic
equilibrium- like process. Inhibition is reversible by substrate. |
Vmax
is unchanged; Km, as defined by [S] required for 1/2 maximal
activity, is increased. |
Noncompetitive Inhibitor |
Binds
E or ES complex other than at the catalytic site. Substrate binding
unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by
substrate. |
Km
appears unaltered; Vmax is decreased proportionately to inhibitor
concentration. |
Uncompetitive Inhibitor |
Binds
only to ES complexes at locations other than the catalytic site. Substrate
binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by
substrate. |
Apparent
Vmax decreased; Km, as defined by [S] required for 1/2
maximal activity, is decreased. |
Inhibitor Type
Specifically at the catalytic site, where it competes with substrate for
binding in a dynamic equilibrium- like process. Inhibition is reversible by
substrate.
Vmax is unchanged; Km, as defined
by [S] required for 1/2 maximal activity, is increased.
http://www.youtube.com/watch?v=0pJOFze055Y
Noncompetitive Inhibitor
Binds E or
ES complex other than at the catalytic site. Substrate binding unaltered, but
ESI complex cannot form products. Inhibition cannot be reversed by substrate.
Km appears
unaltered; Vmax is decreased proportionately to inhibitor concentration.
Uncompetitive Inhibitor
Binds only
to ES complexes at locations other than the catalytic site. Substrate binding
modifies enzyme structure, making inhibitor- binding site available. Inhibition
cannot be reversed by substrate.
Apparent Vmax decreased; Km, as
defined by [S] required for 1/2 maximal activity, is decreased.
The hallmark of all the reversible inhibitors is
that when the inhibitor concentration drops, enzyme activity is regenerated.
Usually these inhibitors bind to enzymes by non-covalent forces and the
inhibitor maintains a reversible equilibrium with the enzyme. The equilibrium
constant for the dissociation of enzyme inhibitor complexes is known as
KI:
KI = [E][I]/[E--I--complex]
The importance of KI is that in all enzyme
reactions where substrate, inhibitor and enzyme interact, the normal Km and or
Vmax for substrate enzyme interaction appear to be altered. These changes are a
consequence of the influence of KI on the overall rate equation for the
reaction. The effects of KI are best observed in Lineweaver-Burk plots.
Probably the best known
reversible inhibitors are competitive inhibitors, which always bind at the
catalytic or active site of the enzyme. Most drugs that alter enzyme activity
are of this type. Competitive inhibitors are especially attractive as clinical
modulators of enzyme activity because they offer two routes for the reversal of
enzyme inhibition, while other reversible inhibitors offer only one. First, as
with all kinds of reversible inhibitors, a decreasing concentration of the
inhibitor reverses the equilibrium regenerating active free enzyme. Second,
since substrate and competitive inhibitors both bind at the same site they
compete with one another for binding .
Raising the concentration of substrate (S), while holding the
concentration of inhibitor constant, provides the second route for reversal of
competitive inhibition. The greater the proportion of substrate, the greater
the proportion of enzyme present in competent ES complexes.
As noted earlier, high concentrations of substrate can displace virtually
all competitive inhibitor bound to active sites. Thus, it is apparent that Vmax
should be unchanged by competitive inhibitors. This characteristic of
competitive inhibitors is reflected in the identical vertical-axis intercepts
of Lineweaver-Burk plots, with and without inhibitor.
Since attaining Vmax requires appreciably higher substrate
concentrations in the presence of competitive inhibitor, Km (the substrate
concentration at half maximal velocity) is also higher, as demonstrated by the
differing negative intercepts on the horizontal axis in panel B.
Analogously, panel C
illustrates that noncompetitive inhibitors appear to have no effect on the
intercept at the x-axis implying that noncompetitive inhibitors have no effect
on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not
interfere in the equilibration of enzyme, substrate and ES complexes, the Km's
of Michaelis-Menten type enzymes are not expected to be affected by
noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C.
However, because complexes that contain inhibitor (ESI) are incapable of
progressing to reaction products, the effect of a noncompetitive inhibitor is
to reduce the concentration of ES complexes that can advance to product.
Since Vmax = k2[Etotal], and the concentration of competent Etotal is
diminished by the amount of ESI formed, noncompetitive inhibitors are expected
to decrease Vmax, as illustrated by the y-axis intercepts in panel C.
A corresponding analysis of uncompetitive inhibition
leads to the expectation that these inhibitors should change the apparent
values of Km as well as Vmax. Changing both constants leads to double
reciprocal plots, in which intercepts on the x and y axes are proportionately
changed; this leads to the production of parallel lines in inhibited and
uninhibited reactions.
Inhibition
Competitive inhibitors bind
reversibly to the enzyme, preventing the binding of substrate. On the other
hand, binding of substrate prevents binding of the inhibitor. Substrate and
inhibitor compete for the enzyme.Main article: Enzyme inhibitor
Enzyme reaction rates can be decreased by various
types of enzyme inhibitors.
Reversible inhibitors
In competitive inhibition the
inhibitor binds to the substrate binding site (figure right, top, thus
preventing substrate from binding (EI complex). Often competitive inhibitors
strongly resemble the real substrate of the enzyme. For example, methotrexate
is a competitive inhibitor of the enzyme dihydrofolate reductase, which
catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity
between the structures of folic acid and this drug are shown in the figure to
the right bottom.
Non-competitive inhibition
In order to
do its work, an enzyme must unite - even if ever so briefly - with at least one
of the reactants. In most cases, the forces that hold the enzyme and its
substrate are noncovalent, an assortment of:
· ionic interactions and hydrophobic
interactions
Link
to discussion of the noncovalent forces that hold macromolecules
Most of these interactions are weak and especially so if the atoms
involved are farther than about one angstrom from each other. So
successful binding of enzyme and substrate requires that the two molecules be
able to approach each other closely over a fairly broad surface. Thus the
analogy that a substrate molecule binds its enzyme like a key in a lock.
This
requirement for complementarity in the configuration of substrate and enzyme
explains the remarkable specificity of most enzymes. Generally, a given
enzyme is able to catalyze only a single chemical reaction or, at most, a few
reactions involving substrates sharing the same general structure.
The necessity for a
close, if brief, fit between enzyme and substrate explains the phenomenon of
competitive inhibition.
It catalyzes the oxidation (by the removal of two
hydrogen atoms) of succinic acid (a). If one adds malonic acid to
cells, or to a test tube mixture of succinic acid and the enzyme, the action of
the enzyme is strongly inhibited. This is because the structure of malonic acid
allows it to bind to the same site on the enzyme (b). But there is no
oxidation so no speedy release of products. The inhibition is called
competitive because if you increase the ratio of succinic to malonic acid in
the mixture, you will gradually restore the rate of catalysis. At a 50:1 ratio,
the two molecules compete on roughly equal terms for the binding (=catalytic)
site on the enzyme.
Non-competitive inhibitors can bind either to the
active site, or to other parts of the enzyme far away from the
substrate-binding site. Moreover, non-competitive inhibitors bind to the
enzyme-substrate (ES) complex and to the free enzyme. Their binding to this
site changes the shape of the enzyme and stops the active site binding
substrate(s). Consequently, since there is no direct competition between the
substrate and inhibitor for the enzyme, the extent of inhibition depends only
on the inhibitor concentration and will not be affected by the substrate
concentration.