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 220.127.116.11 and 18.104.22.168, the two citrate hydro-lyases, EC 22.214.171.124 and 126.96.36.199, and the two amine oxidases, EC 188.8.131.52 and 184.108.40.206. Others are mainly historical, e.g. acid and alkaline phosphatases (EC 220.127.116.11 and EC 18.104.22.168).
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 22.214.171.124, 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.
Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.).
Act on many chemical groupings to add or remove hydrogen atoms.
Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.
Add water across a bond, hydrolyzing it.
Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds.
Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.
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.
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 126.96.36.199, now believed to be identical with EC 188.8.131.52). 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 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 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 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 184.108.40.206) and cystathionine β-synthase (EC 220.127.116.11).
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).
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 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 18.104.22.168 and EC 22.214.171.124).
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
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 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,
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,
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.
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.
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.
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.
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.
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 → 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.
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 heat labile ALP is stable at
-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.
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.
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.
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.
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
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.
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.
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:
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.
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.
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
Enzymes combine briefly with reactants during an enzyme-catalyzed reaction.
Enzymes are released unchanged after catalyzing the conversion of reactants to Product
Enzymes are specific in their activity; each enzyme catalyzes the reaction of a single type of molecules or a group of closely related molecules.
Enzymes are saturated by high substrate concentrations.
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.
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).
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.
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.
· 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.
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.
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.
· 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.
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).
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.
· 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 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.
Å. coli also has a mechanism which regulates enzyme synthesis by controlling translation of a needed messenger RNA..
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.
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.
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.
Binding Site on Enzyme
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.
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.
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.
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.
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.
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.
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.
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.
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:
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.