Isozyme

Inbiochemistry,isozymes(also known asisoenzymesor more generally asmultiple forms of enzymes) areenzymesthat differ in amino acid sequence but catalyze the same chemical reaction. Isozymes usually have different kinetic parameters (e.g. differentK_Mvalues), 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 byhomologousgenes 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, orallozymesif encoded by differentallelesof the samegene;the two terms are often used interchangeably.

Introduction

Isozymes were first described byR. L. HunterandClement Markert(1957) who defined them asdifferent 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 differentloci(described asisozymes) and (2) enzymes that are the product of differentallelesof the same gene (described asallozymes).

Isozymes are usually the result ofgene duplication,but can also arise frompolyploidisationornucleic acid hybridization.Over evolutionary time, if the function of the new variant remainsidenticalto the original, then it is likely that one or the other will be lost asmutationsaccumulate, resulting in apseudogene.However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern ofexpression,then the two variants may both be favoured bynatural selectionand become specialised to different functions.For example, they may be expressed at different stages of development or in different tissues.

Allozymes may result frompoint mutationsor from insertion-deletion (indel) 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 lowfitnessand be removed from the population bynatural selection.
• Alternatively, if theamino acidresidue that is changed is in a relatively unimportant part of the enzyme (e.g., a long way from theactive site), then the mutation may beselectively neutraland subject togenetic drift.
• In rare cases, the mutation may result in an enzyme that is more efficient, or one that can catalyse a slightly differentchemical reaction,in which case the mutation may cause an increase in fitness, and be favoured by natural selection.

Examples

An example of an isozyme isglucokinase,a variant ofhexokinasewhich is not inhibited byglucose 6-phosphate.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 ofinsulinrelease by thebeta cellsof thepancreas,or initiation ofglycogensynthesis bylivercells. Both these processes must only occur when glucose is abundant.

The 5 isozymes of LDH

Distinction between five isozymes usingelectrophoresis

1.) The enzymelactate dehydrogenaseis a tetramer made of two different sub-units, the H-form and the M-form. These combine in differentcombinationsdepending on the tissue:

Type	Composition	Location	Electrophoretic Mobility	Whether destroyed by Heat (at 60 °C)	Percentage of normal serum in humans
LDH1	HHHH	Heart and Erythrocyte	Fastest	No	25%
LDH2	HHHM	Heart and Erythrocyte	Faster	No	35%
LDH3	HHMM	Brain and Kidney	Fast	Partially	27%
LDH4	HMMM	Skeletal Muscle and Liver	Slow	Yes	8%
LDH5	MMMM	Skeletal Muscle and Liver	Slowest	Yes	5%

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

Isoenzyme	Subunit	Tissue of Origin
CPK1	BB	Brain
CPK2	MB	Heart
CPK3	MM	Skeletal muscle

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 theirsubstratesandenzyme kinetics,they may be distinguished by abiochemical assay.However, such differences are usually subtle, particularly betweenallozymeswhich are oftenneutral variants.This subtlety is to be expected, because two enzymes that differ significantly in their function are unlikely to have been identified asisozymes.

While isozymes may be almost identical in function, they may differ in other ways. In particular,amino acidsubstitutions that change theelectric chargeof the enzyme are simple to identify bygel electrophoresis,and this forms the basis for the use of isozymes asmolecular markers.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 frompotato starch,butacrylamidegels 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 localisedprecipitationof soluble indicatordyessuch astetrazolium saltswhich become insoluble when they arereducedbycofactorssuch asNADorNADP,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 differentK_Mand V_maxvalues).

Isozymes and allozymes as molecular markers

Population geneticsis 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 usedmolecular markersfor this purpose. Although they have now been largely superseded by more informativeDNA-based approaches (such as directDNA sequencing,single nucleotide polymorphismsandmicrosatellites), they are still among 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. quantifyingmating systems.

Other major examples

• Thecytochrome P450isozymes play important roles inmetabolismandsteroidogenesis.
• The multiple forms ofphosphodiesterasealso 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

• Hunter, R. L.; Merkert, C.L. (1957). "Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels".Science.125(3261): 1294–1295.doi:10.1126/science.125.3261.1294-a.PMID13432800.
• Weiss, B.; Hait, W.N. (1977). "Selective cyclic nucleotide phosphodiesterase inhibitors as potential therapeutic agents".Annu. Rev. Pharmacol. Toxicol.17:441–477.doi:10.1146/annurev.pa.17.040177.002301.PMID17360.
• Wendel, JF, and NF Weeden. 1990. "Visualisation and interpretation of plant isozymes." pp. 5–45 inD. E. SoltisandP. S. Soltis,eds.Isozymes in plant biology.Chapman and Hall, London.
• Weeden, NF, and JF Wendel. 1990. "Genetics of plant isozymes". pp. 46–72 inD. E. SoltisandP. S. Soltis,eds.Isozymes in plant biology.Chapman and Hall, London
• Crawford, DJ. 1989. "Enzyme electrophoresis and plant systematics". pp. 146–164 inD. E. SoltisandP. 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)