Editing Isozyme

{{Short description|Enzymes that differ in amino acid sequence but catalyze the same chemical reaction}}
In [[biochemistry]], '''isozymes''' (also known as '''isoenzymes''' or more generally as '''multiple forms of enzymes''') are [[enzyme]]s that differ in amino acid sequence but catalyze the same chemical reaction.  Isozymes usually have different kinetic parameters (e.g. different [[Michaelis-Menten kinetics|''K''<sub>M</sub>]] 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 [[homology (biology)|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]]s if encoded by different [[allele]]s of the same [[gene]]; the two terms are often used interchangeably.

==Introduction==
Isozymes 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''.<ref>{{Cite journal|last1=Markert|first1=Clement L.|last2=Moller|first2=Freddy|year=1959|title=Multiple forms of enzymes: tissue, ontogenetic, and species specific patterns.|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=45|issue=5|pages=753–763|doi=10.1073/pnas.45.5.753|pmc=222630|pmid=16590440|doi-access=free}}</ref>  This definition encompasses (1) enzyme variants that are the product of different genes and thus represent different [[locus (genetics)|loci]] (described as ''isozymes'') and (2) enzymes that are the product of different [[alleles]] of the same gene (described as ''allozymes'').<ref name="Kearney">{{cite book |last1=Kearney |title=Fundamental Genetics |date=2014 |publisher=McNaughton Publishing |pages=413–414 |edition=3rd}}</ref>

Isozymes are usually the result of [[gene duplication]], but can also arise from [[polyploidy|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 [[mutation]]s 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|expression]], then the two variants may both be favoured by [[natural selection]] and become specialised to different functions.<ref>{{cite book |last1=Gerald, Gerald |title=The Biology Book: From the Origin of Life to Epigenetics, 250 Milestones in the History of Biology |date=2015 |publisher=Sterling |page=79}}</ref> For example, they may be expressed at different stages of development or in different tissues.<ref name="Huang">{{cite book |last1=Huang, Le |title=Genome |date=2009 |publisher=Grady-McPherson |page=299}}</ref>

Allozymes may result from [[point mutation]]s or from insertion-deletion ([[mutation|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 low [[fitness (biology)|fitness]] and be removed from the population by [[natural selection]].<ref name="Alberts">{{cite book |last1=Alberts |title=Molecular Biology of the Cell |date=2017 |publisher=Garland Science |page=649 |edition=6th}}</ref>
* 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 [[Neutral theory of molecular evolution|selectively neutral]] and subject to [[genetic drift]].<ref name="Walstrom">{{cite journal |last1=Walstrom, Ford|display-authors=etal |title=Models of genetics and natural selection: a current biomolecular understanding |journal=Biomolecular Ecology |date=2014 |volume=70 |issue=2 |pages=1021–1034}}</ref>
* 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.<ref name="Walstrom" />

==Examples==
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), allow it to serve different functions in cells of specific organs, such as control of [[insulin]] release by the [[beta cell]]s of the [[pancreas]], or initiation of [[glycogen]] synthesis by [[liver]] cells. Both these processes must only occur when glucose is abundant.

[[File:Isoenzyme1.jpg|thumb|The 5 isozymes of LDH]]
[[File:Isoenzyme2.jpg|thumb|right|Distinction between five isozymes using [[electrophoresis]]]]
1.) The enzyme [[lactate dehydrogenase]] is a tetramer made of two different sub-units, the H-form and the M-form.  These combine in different [[combination]]s depending on the tissue:<ref name=":0">{{Cite book|title=Biochemistry|last=Satyanarayana, U.|date=2002|publisher=Books and Allied|isbn=8187134801|edition=2nd|location=Kolkata, India|oclc=71209231}}</ref>
    {| class="wikitable"
|-
! Type !! Composition !! Location
!Electrophoretic Mobility
!Whether destroyed by 
Heat (at 60&nbsp;°C)
!Percentage of normal 
serum in humans 
|-
| LDH<sub>1</sub> || HHHH || Heart and Erythrocyte
|Fastest
|No
|25%
|-
| LDH<sub>2</sub> || HHHM || Heart and Erythrocyte
|Faster
|No
|35%
|-
| LDH<sub>3</sub> || HHMM || Brain and Kidney
|Fast
|Partially 
|27%
|-
| LDH<sub>4</sub> || HMMM || Skeletal Muscle and Liver
|Slow
|Yes
|8%
|-
| LDH<sub>5</sub> || MMMM || Skeletal Muscle and Liver
|Slowest
|Yes
|5%
|}
2.) Isoenzymes of creatine phosphokinase:<ref name=":0" /> 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<ref name=":0" />
{| class="wikitable"
|+
!Isoenzyme
!Subunit
!Tissue of Origin
|-
|CPK<sub>1</sub>
|BB
|Brain
|-
|CPK<sub>2</sub>
|MB
|Heart 
|-
|CPK<sub>3</sub>
|MM
|Skeletal muscle
|}
3.) Isoenzymes of alkaline phosphatase:<ref name=":0" /> 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 α<sub>1</sub>-ALP, α<sub>2</sub>-heat labile ALP, α<sub>2</sub>-heat stable ALP, pre-β ALP and γ-ALP. Increase in  α<sub>2</sub>-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 [[Substrate (biochemistry)|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 theory of molecular evolution|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]] substitutions that change the [[electric charge]] of the enzyme are simple to identify by [[gel electrophoresis]], and this forms the basis for the use of isozymes as [[molecular marker]]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]], 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 (chemistry)|precipitation]] of soluble indicator [[dye]]s such as [[tetrazolium salts]] which become insoluble when they are [[Redox|reduced]] by [[Cofactor (biochemistry)|cofactors]] such as [[Nicotinamide adenine dinucleotide|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 [[Michaelis-Menten kinetics|''K''<sub>M</sub> and V<sub>max</sub>]] values).

==Isozymes and allozymes as molecular markers ==
[[Population genetics]] 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]]s for this purpose. Although they have now been largely superseded by more informative [[DNA]]-based approaches (such as direct [[DNA sequencing]], [[single nucleotide polymorphism]]s and [[Microsatellite (genetics)|microsatellites]]), they are still among the quickest and cheapest marker systems to develop, and remain ({{As of|2005|lc=on}}) an excellent choice for projects that only need to identify low levels of genetic variation, e.g. quantifying [[mating system]]s.

==Other major examples==
*The [[cytochrome P450]] isozymes play important roles in [[metabolism]] and [[steroidogenesis]].
*The multiple forms of [[phosphodiesterase]] 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 = Hunter | first1 = R. L. | last2 = Merkert | first2 = C.L. | year = 1957 | title = Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels | journal = Science | volume = 125 | issue = 3261 | pages = 1294–1295 | doi=10.1126/science.125.3261.1294-a| pmid = 13432800 }}
* {{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.&nbsp;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.&nbsp;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.&nbsp;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.&nbsp;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
<references />

==External links==
* [http://www.genetics.cf.adfg.state.ak.us/techfac/electro2.php Allozyme Electrophoresis Techniques] – a complete guide to starch gel electrophoresis
* [http://www.chem.ttu.ee/teadus/teema_en?t=T499 Development of new isozyme specific therapeutics] – Fatty Acid Dioxygenases and Eicosanoid Hormones (Estonia)

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[[Category:Enzymes]]
[[Category:Biochemistry]]