ISOENZYMES. ENZYMODIAGNOSTICS. ENZYMOPATHY. ENZYMOTHERAPY
Isoenzymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties. The existence of isoenzymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (for example lactate dehydrogenase (LDH)). In biochemistry, isoenzymes are isoforms (closely related variants) of enzymes. In many cases, they are coded for by homologous genes that have diverged over time. Although, strictly speaking, allozymes represent enzymes from different alleles of the same gene, and isoenzymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.
Isoenzymes were first described by R. L. Hunter and Clement Markert (1957) who defined them as different variants of the same enzyme having identical functions and present in the same individual. This definition encompasses (1) enzyme variants that are the product of different genes and thus represent different loci (described as isoenzymes) and (2) enzymes that are the product of different alleles of the same gene (described as allozymes).
Isoenzymes are usually the result of gene duplication, but can also arise from polyploidisation or nucleic acid hybridization. Over evolutionary time, if the function of the new variant remains identical to the original, then it is likely that one or the other will be lost as mutations accumulate, resulting in a pseudogene. However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern of gene expression, then the two variants may both be favoured by natural selection and become specialised to different functions. For example, they may be expressed at different stages of development or in different tissues.
Allozymes may result from point mutations or from insertion-deletion (indel) events that affect the DNA coding sequence of the gene. As with any other new mutation, there are three things that may happen to a new allozyme:
It is most likely that the new allele will be non-functional — in which case it will probably result in low fitness and be removed from the population by natural selection.
Alternatively, if the amino acid residue that is changed is in a relatively unimportant part of the enzyme (e.g., a long way from the active site), then the mutation may be selectively neutral and subject to genetic drift.
In rare cases, the mutation may result in an enzyme that is more efficient, or one that can catalyse a slightly different chemical reaction, in which case the mutation may cause an increase in fitness, and be favoured by natural selection.
An example of an isozyme is glucokinase, a variant of hexokinase which is not inhibited by glucose 6-phosphate. Its different regulatory features and lower affinity for glucose (compared to other hexokinases), allows it to serve different functions in cells of specific organs, such as control of insulin release by the beta cells of the pancreas, or initiation of glycogen synthesis by liver cells. Both of these processes must only occur when glucose is abundant, or problems occur.
The enzyme Lactate Dehydrogenase is made of two(H-form and M-Form) different sub units, combines in different Permutations and Combinations in depending on the tissue in which it is present as shown in below table,Type
Isoenzymes (and allozymes) are variants of the same enzyme. Unless they are identical in terms of their biochemical properties, for example their substrates and enzyme kinetics, they may be distinguished by a biochemical assay. However, such differences are usually subtle (particularly between allozymes which are often neutral variants). This subtlety is to be expected, because two enzymes that differ significantly in their function are unlikely to have been identified as isoenzymes.
Whilst isoenzymes may be almost identical in function, they may differ in other ways. In particular, amino acid substitutions that change the electric charge of the enzyme (such as replacing aspartic acid with glutamic acid) are simple to identify by gel electrophoresis, and this forms the basis for the use of isoenzymes as molecular markers. To identify isoenzymes, a crude protein extract is made by grinding animal or plant tissue with an extraction buffer, and the components of extract are separated according to their charge by gel electrophoresis. Historically, this has usually been done using gels made from potato starch, but acrylamide gels provide better resolution.
All the proteins from the tissue are present in the gel, so that individual enzymes must be identified using an assay that links their function to a staining reaction. For example, detection can be based on the localised precipitation of soluble indicator dyes such as tetrazolium salts which become insoluble when they are reduced by cofactors such as NAD or NADP, which generated in zones of enzyme activity. This assay method requires that the enzymes are still functional after separation (native gel electrophoresis), and provides the greatest challenge to using isoenzymes as a laboratory technique.
Isoenzymes differ in kinetics (they have different Km and Vmax values.
Population genetics is essentially a study of the causes and effects of genetic variation within and between populations, and in the past, isoenzymes have been amongst the most widely used molecular markers for this purpose. Although they have now been largely superseded by more informative DNA-based approaches (such as direct DNA sequencing, single nucleotide polymorphisms and microsatellites), they are still amongst the quickest and cheapest marker systems to develop, and remain (as of 2005) an excellent choice for projects that only need to identify low levels of genetic variation, e.g. quantifying mating systems.
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
Although enzymes are highly specific for the kind of reaction they catalyze, the same is not always true of substrates they attack. For example, while succinic dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid, alcohol dehydrogenase (ADH) always catalyzes oxidation-reduction reactions but attacks a number of different alcohols, ranging from methanol to butanol. Generally, enzymes having broad substrate specificity are most active against one particular substrate. In the case of ADH, ethanol is the preferred substrate.
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 isoenzymes. These are the products of genes that vary only slightly; often, various isoenzymes of a group are expressed in different tissues of the body. The best studied set of isoenzymes 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 isoenzymes. 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 isoenzymes all catalyze the same chemical reaction, but they exhibit differing degrees of efficiency. The detection of specific LDH isoenzymes in the blood is highly diagnostic of tissue damage such as occurs during cardiac infarct.
Non-competitive inhibition
In order to do its work, an enzyme must unite - even if ever so briefly - with at least one of the reactants. In most cases, the forces that hold the enzyme and its substrate are noncovalent, an assortment of:
• hydrogen bonds
• ionic interactions
• and hydrophobic interactions
Link to discussion of the noncovalent forces that hold macromolecules
together.
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.
Biological function
Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies.
Viruses can contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.
An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch is inabsorbable in the intestine but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants which have a herbivorous diets, bacteria in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.
Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyse the same reaction in parallel, this can allow more complex regulation: with for example a low constant activity being provided by one enzyme but an inducible high activity from a second enzyme.
Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.
Control activity
There are five main ways that enzyme activity is controlled in the cell.
Regulation of Enzyme Activity
While it is clear that enzymes are responsible for the catalysis of almost all biochemical reactions, it is important to also recognize that rarely, if ever, do enzymatic reactions proceed in isolation. The most common scenario is that enzymes catalyze individual steps of multi-step metabolic pathways, as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a consequence of these lock- step sequences of reactions, any given enzyme is dependent on the activity of preceding reaction steps for its substrate.
In humans, substrate concentration is dependent on food supply and is not usually a physiologically important mechanism for the routine regulation of enzyme activity. Enzyme concentration, by contrast, is continually modulated in response to physiological needs. Three principal mechanisms are known to regulate the concentration of active enzyme in tissues:
1. Regulation of gene expression controls the quantity and rate of enzyme synthesis.
2. Proteolytic enzyme activity determines the rate of enzyme degradation.
3. Covalent modification of preexisting pools of inactive proenzymes produces active enzymes.
Enzyme synthesis and proteolytic degradation are comparatively slow mechanisms for regulating enzyme concentration, with response times of hours, days or even weeks. Proenzyme activation is a more rapid method of increasing enzyme activity but, as a regulatory mechanism, it has the disadvantage of not being a reversible process. Proenzymes are generally synthesized in abundance, stored in secretory granules and covalently activated upon release from their storage sites. Examples of important proenzymes include pepsinogen, trypsinogen and chymotrypsinogen, which give rise to the proteolytic digestive enzymes. Likewise, many of the proteins involved in the cascade of chemical reactions responsible for blood clotting are synthesized as proenzymes. Other important proteins, such as peptide hormones and collagen, are also derived by covalent modification of precursors.
Another mechanism of regulating enzyme activity is to sequester enzymes in compartments where access to their substrates is limited. For example, the proteolysis of cell proteins and glycolipids by enzymes responsible for their degradation is controlled by sequestering these enzymes within the lysosome.
In contrast to regulatory mechanisms that alter enzyme concentration, there is an important group of regulatory mechanisms that do not affect enzyme concentration, are reversible and rapid in action, and actually carry out most of the moment- to- moment physiological regulation of enzyme activity. These mechanisms include allosteric regulation, regulation by reversible covalent modification and regulation by control proteins such as calmodulin. Reversible covalent modification is a major mechanism for the rapid and transient regulation of enzyme activity. The best examples, again, come from studies on the regulation of glycogen metabolism where phosphorylation of glycogen synthase and glycogen phosphorylase kinase results in the stimulation of glycogen degradation while glycogen synthesis is coordinately inhibited. Numerous other enzymes of intermediary metabolism are affected by phosphorylation, either positively or negatively. These covalent phosphorylations can be reversed by a separate sub-subclass of enzymes known as phosphatases. Recent research has indicated that the aberrant phosphorylation of growth factor and hormone receptors, as well as of proteins that regulate cell division, often leads to unregulated cell growth or cancer. The usual sites for phosphate addition to proteins are the serine, threonine and tyrosine R group hydroxyl residues.
Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions.
Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.
Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.
Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen.
Some enzymes may become activated when localized to a different environment (eg. from a reducing (cytoplasm) to an oxidising (periplasm) environment, high pH to low pH etc). For example, hemagglutinin of the influenza virus undergoes a conformational change once it encounters the acidic environment of the host cell vesicle causing its activation.
Involvement in disease Phenylalanine hydroxylase. Created from PDB 1KW0Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The importance of enzymes is shown by the fact that a lethal illness can be caused by the malfunction of just one type of enzyme out of the thousands of types present in our bodies.
One example is the most common type of phenylketonuria. A mutation of a single amino acid in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, results in build-up of phenylalanine and related products. This can lead to mental retardation if the disease is untreated.
Another example is when germline mutations in genes coding for DNA repair enzymes cause hereditary cancer syndromes such as xeroderma pigmentosum. Defects in these enzymes cause cancer since the body is less able to repair mutations in the genome. This causes a slow accumulation of mutations and results in the development of many types of cancer in the sufferer.
Naming conventions
An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are lactase, alcohol dehydrogenase and DNA polymerase. This may result in different enzymes, called isoenzymes, with the same function having the same basic name. Isoenzymes have a different amino acid sequence and might be distinguished by their optimal pH, kinetic properties or immunologically. Furthermore, the normal physiological reaction an enzyme catalyzes may not be the same as under artifical conditions. This can result in the same enzyme being identified with two different names. E.g. Glucose isomerase, used industrially to convert glucose into the sweetener fructose, is a xylose isomerase in vivo.
Allosteric Enzymes
Allosteric modulation
Allosteric enzymes change their structure in response to binding of effectors. Modulation can be direct, where the effector binds directly to binding sites in the enzyme, or indirect, where the effector binds to other proteins or protein subunits that interact with the allosteric enzyme and thus influence catalytic activity.
In addition to simple enzymes that interact only with substrates and inhibitors, there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).
The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme's active site. Those that increase catalytic activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.
Most allosteric enzymes are oligomeric (consisting of multiple subunits); generally they are located at or near branch points in metabolic pathways, where they are influential in directing substrates along one or another of the available metabolic paths. The effectors that modulate the activity of these allosteric enzymes are of two types. Those activating and inhibiting effectors that bind at allosteric sites are called heterotropic effectors. (Thus there exist both positive and negative heterotropic effectors.) These effectors can assume a vast diversity of chemical forms, ranging from simple inorganic molecules to complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single defining feature is that they are not identical to the substrate.
In many cases the substrate itself induces distant allosteric effects when it binds to the catalytic site. Substrates acting as effectors are said to be homotropic effectors. When the substrate is the effector, it can act as such, either by binding to the substrate-binding site, or to an allosteric effector site. When the substrate binds to the catalytic site it transmits an activity-modulating effect to other subunits of the molecule. Often used as the model of a homotropic effector is hemoglobin, although it is not a branch-point enzyme and thus does not fit the definition on all counts.
There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered. Enzymes whose Km is altered by effectors are said to be K-type enzymes and the effector a K-type effector. If Vmax is altered, the enzyme and effector are said to be V-type. Many allosteric enzymes respond to multiple effectors with V-type and K-type behavior. Here again, hemoglobin is often used as a model to study allosteric interactions, although it is not strictly an enzyme.