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biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology an ...
, isozymes (also known as isoenzymes or more generally as multiple forms of enzymes) are
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. ...
s that differ in amino acid sequence but catalyze the same chemical reaction. Isozymes usually have different kinetic parameters (e.g. different ''K''M values), or are regulated differently. They permit the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage. In many cases, isozymes are encoded by homologous genes that have diverged over time. Strictly speaking, enzymes with different amino acid sequences that catalyse the same reaction are isozymes if encoded by different genes, or
allozyme Alloenzymes (or also called allozymes) are variant forms of an enzyme which differ structurally but not functionally from other allozymes coded for by different alleles at the same locus. These are opposed to isozymes, which are enzymes that perf ...
s if encoded by different
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chro ...
s of the same
gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ...
; the two terms are often used interchangeably.


Introduction

Isozymes were first described by R. L. Hunter and
Clement Markert Clement Lawrence Markert (April 11, 1917 – October 1, 1999) was an American biologist credited with the discovery of isozymes (different forms of enzymes that catalyze the same reaction). He was a member of the National Academy of Sciences ...
(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 ''isozymes'') and (2) enzymes that are the product of different
alleles An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chr ...
of the same gene (described as ''allozymes''). Isozymes are usually the result of
gene duplication Gene duplication (or chromosomal duplication or gene amplification) is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gen ...
, but can also arise from polyploidisation or
nucleic acid hybridization Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucle ...
. 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
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...
s accumulate, resulting in a
pseudogene Pseudogenes are nonfunctional segments of DNA that resemble functional genes. Most arise as superfluous copies of functional genes, either directly by DNA duplication or indirectly by reverse transcription of an mRNA transcript. Pseudogenes are ...
. However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern of
expression Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Fixed expression, a form of words with a specific meaning * Idiom, a type of fixed expression * Metaphorical expression, a particular word, phrase, o ...
, then the two variants may both be favoured by
natural selection Natural selection is the differential survival and reproduction of individuals due to differences in phenotype. It is a key mechanism of evolution, the change in the heritable traits characteristic of a population over generations. Char ...
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 mutation A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...
s or from insertion-deletion (
indel Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism. It is classified among small genetic variations, measuring from 1 to 10 000 base pairs in length, including insertion and deletion events that ...
) events that affect the coding sequence of the gene. As with any other new mutations, 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 Natural selection is the differential survival and reproduction of individuals due to differences in phenotype. It is a key mechanism of evolution, the change in the heritable traits characteristic of a population over generations. Char ...
. * Alternatively, if the
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
residue that is changed is in a relatively unimportant part of the enzyme (e.g., a long way from the
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) ...
), then the mutation may be selectively neutral and subject to
genetic drift Genetic drift, also known as allelic drift or the Wright effect, is the change in the frequency of an existing gene variant (allele) in a population due to random chance. Genetic drift may cause gene variants to disappear completely and there ...
. * In rare cases, the mutation may result in an enzyme that is more efficient, or one that can catalyse a slightly different
chemical reaction A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the positions of electrons in the forming and breakin ...
, in which case the mutation may cause an increase in fitness, and be favoured by natural selection.


Examples

An example of an isozyme is
glucokinase Glucokinase () is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver and pancreas of humans and most other vertebrates. In each of these organs it plays an important role in t ...
, a variant of
hexokinase A hexokinase is an enzyme that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate. In most organisms, glucose is the most important substrate for hexokinases, and glucose-6-phosphate is the most important product. Hexoki ...
which is not inhibited by
glucose 6-phosphate Glucose 6-phosphate (G6P, sometimes called the Robison ester) is a glucose sugar phosphorylated at the hydroxy group on carbon 6. This dianion is very common in cells as the majority of glucose entering a cell will become phosphorylated in this way ...
. Its different regulatory features and lower affinity for glucose (compared to other hexokinases), allow it to serve different functions in cells of specific organs, such as control of
insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolis ...
release by the
beta cell Beta cells (β-cells) are a type of cell found in pancreatic islets that synthesize and secrete insulin and amylin. Beta cells make up 50–70% of the cells in human islets. In patients with Type 1 diabetes, beta-cell mass and function are dimini ...
s of the
pancreas The pancreas is an organ of the digestive system and endocrine system of vertebrates. In humans, it is located in the abdomen behind the stomach and functions as a gland. The pancreas is a mixed or heterocrine gland, i.e. it has both an end ...
, or initiation of
glycogen Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body. Glycogen functions as one o ...
synthesis by
liver The liver is a major organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the synthesis of proteins and biochemicals necessary for digestion and growth. In humans, it ...
cells. Both these processes must only occur when glucose is abundant. 1.) The enzyme
lactate dehydrogenase Lactate dehydrogenase (LDH or LD) is an enzyme found in nearly all living cells. LDH catalyzes the conversion of lactate to pyruvate and back, as it converts NAD+ to NADH and back. A dehydrogenase is an enzyme that transfers a hydride from on ...
is a tetramer made of two different sub-units, the H-form and the M-form. These combine in different
combination In mathematics, a combination is a selection of items from a set that has distinct members, such that the order of selection does not matter (unlike permutations). For example, given three fruits, say an apple, an orange and a pear, there are th ...
s depending on the tissue: 2.) Isoenzymes of creatine phosphokinase: Creatine kinase (CK) or creatine phosphokinase (CPK) catalyses the interconversion of phospho creatine to creatine . CPK exists in 3 isoenzymes. Each isoenzymes is a dimer of 2 subunits M (muscle), B (brain) or both 3.) Isoenzymes of alkaline phosphatase: Six isoenzymes have been identified. The enzyme is a monomer, the isoenzymes are due to the differences in the carbohydrate content (sialic acid residues). The most important ALP isoenzymes are α1-ALP, α2-heat labile ALP, α2-heat stable ALP, pre-β ALP and γ-ALP. Increase in α2-heat labile ALP suggests hepatitis whereas pre-β ALP indicates bone diseases.


Distinguishing isozymes

Isozymes (and allozymes) are variants of the same enzyme. Unless they are identical in their biochemical properties, for example their substrates and
enzyme kinetics Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in ...
, 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 ''isozymes''. While isozymes may be almost identical in function, they may differ in other ways. In particular,
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
substitutions that change the
electric charge Electric charge is the physical property of matter that causes charged matter to experience a force when placed in an electromagnetic field. Electric charge can be ''positive'' or ''negative'' (commonly carried by protons and electrons respecti ...
of the enzyme are simple to identify by
gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
, and this forms the basis for the use of isozymes as
molecular marker A molecular marker is a molecule, sampled from some source, that gives information about its source. For example, DNA is a molecular marker that gives information about the organism from which it was taken. For another example, some proteins can b ...
s. To identify isozymes, 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 Potato starch is starch extracted from potatoes. The cells of the root tubers of the potato plant contain leucoplasts (starch grains). To extract the starch, the potatoes are crushed, and the starch grains are released from the destroyed cells. Th ...
, but
acrylamide Acrylamide (or acrylic amide) is an organic compound with the chemical formula CH2=CHC(O)NH2. It is a white odorless solid, soluble in water and several organic solvents. From the chemistry perspective, acrylamide is a vinyl-substituted prima ...
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 In meteorology, precipitation is any product of the condensation of atmospheric water vapor that falls under gravitational pull from clouds. The main forms of precipitation include drizzle, rain, sleet, snow, ice pellets, graupel and hail. ...
of soluble indicator
dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution a ...
s such as tetrazolium salts which become insoluble when they are reduced by
cofactors Cofactor may also refer to: * Cofactor (biochemistry), a substance that needs to be present in addition to an enzyme for a certain reaction to be catalysed * A domain parameter in elliptic curve cryptography, defined as the ratio between the orde ...
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 isozymes as a laboratory technique. Isoenzymes differ in kinetics (they have different ''K''M and Vmax values).


Isozymes and allozymes as molecular markers

Population genetics Population genetics is a subfield of genetics that deals with genetic differences within and between populations, and is a part of evolutionary biology. Studies in this branch of biology examine such phenomena as adaptation, speciation, and po ...
is essentially a study of the causes and effects of genetic variation within and between populations, and in the past, isozymes have been amongst the most widely used
molecular marker A molecular marker is a molecule, sampled from some source, that gives information about its source. For example, DNA is a molecular marker that gives information about the organism from which it was taken. For another example, some proteins can b ...
s for this purpose. Although they have now been largely superseded by more informative DNA-based approaches (such as direct
DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. T ...
,
single nucleotide polymorphism In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
s and microsatellites), they are still among the quickest and cheapest marker systems to develop, and remain () an excellent choice for projects that only need to identify low levels of genetic variation, e.g. quantifying
mating system A mating system is a way in which a group is structured in relation to sexual behaviour. The precise meaning depends upon the context. With respect to animals, the term describes which males and females mate under which circumstances. Recognised s ...
s.


Other major examples

*The
cytochrome P450 Cytochromes P450 (CYPs) are a superfamily of enzymes containing heme as a cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various com ...
isozymes play important roles in
metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run ...
and
steroidogenesis A steroid is a biologically active organic compound with four rings arranged in a specific molecular configuration. Steroids have two principal biological functions: as important components of cell membranes that alter membrane fluidity; and ...
. *The multiple forms of
phosphodiesterase A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond. Usually, ''phosphodiesterase'' refers to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below. However, there are many ...
also play major roles in various biological processes. Although more than one form of these enzymes have been found in individual cells, these isoforms of the enzyme are unequally distributed in the various cells of an organism. From the clinical standpoint they have been found to be selectively activated and inhibited, an observation which has led to their use in therapy.


References

* * {{cite journal , last1 = Weiss , first1 = B. , last2 = Hait , first2 = W.N. , year = 1977 , title = Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents , journal = Annu. Rev. Pharmacol. Toxicol. , volume = 17 , pages = 441–477 , doi=10.1146/annurev.pa.17.040177.002301 , pmid = 17360 * Wendel, JF, and NF Weeden. 1990. "Visualisation and interpretation of plant isozymes." pp. 5–45 in D. E. Soltis and P. S. Soltis, eds. ''Isozymes in plant biology.'' Chapman and Hall, London. * Weeden, NF, and JF Wendel. 1990. "Genetics of plant isozymes". pp. 46–72 in D. E. Soltis and P. S. Soltis, eds. ''Isozymes in plant biology.'' Chapman and Hall, London * Crawford, DJ. 1989. "Enzyme electrophoresis and plant systematics". pp. 146–164 in D. E. Soltis and P. S. Soltis, eds. ''Isozymes in plant biology.'' Dioscorides, Portland, Oregon. *Hamrick, JL, and MJW Godt. 1990. "Allozyme diversity in plant species". pp. 43–63 in A. H. D. Brown, M. T. Clegg, A. L. Kahler and B. S. Weir, eds. ''Plant Population Genetics, Breeding, and Genetic Resources.'' Sinauer, Sunderland *Biochemistry by jeremy M. Berg, John L. Tymoczko, Lubert Stryer (Intro taken from this textbook) ;Specific


External links


Allozyme Electrophoresis Techniques
– a complete guide to starch gel electrophoresis
Development of new isozyme specific therapeutics
– Fatty Acid Dioxygenases and Eicosanoid Hormones (Estonia) Enzymes Biochemistry