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In
biochemistry Biochemistry or biological chemistry, is the study of chemical process In a scientific Science () is a systematic enterprise that Scientific method, builds and organizes knowledge in the form of Testability, testable explanations and pr ...

biochemistry
, isozymes (also known as isoenzymes or more generally as multiple forms of enzymes) are
enzyme Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates in ...

enzyme
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 Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substr ...
s if encoded by different
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is one of two, or more, forms of a given gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendelian_inheritance ...
s of the same
gene In biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mecha ...

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''. This definition encompasses (1) enzyme variants that are the product of different genes and thus represent different
loci Locus (plural loci) is Latin for "place". It may refer to: Entertainment * Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front * Locus (magazine), ''Locus'' (magazine), science fiction and fantasy magazine ...
(described as ''isozymes'') and (2) enzymes that are the product of different
alleles An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is one of two, or more, forms of a given gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendelian_inheritance ...

alleles
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 Molecular evolution is the process of change in the sequence composition of c ...
, but can also arise from polyploidisation or
nucleic acid hybridization In molecular biology, hybridization (or hybridisation) is a phenomenon in which single-stranded deoxyribonucleic acid ( DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Though a double-stranded DNA sequence is generally ...
. 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 Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, Biochemistry, chemical processes, Molecular biology, molecular interactions, Physiology, physiological mechan ...
s accumulate, resulting in a
pseudogene Pseudogenes are nonfunctional segments of DNA The structure of part of a DNA double helix Deoxyribonucleic acid (; DNA) is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, wh ...
. However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern of
expression
expression
, 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 right , Here the relation between genotype and phenotype is illustrated, using a Punnett square, for the character of peta ...
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 or substitution 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 ...

point mutation
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 right , Here the relation between genotype and phenotype is illustrated, using a Punnett square, for the character of peta ...
. * Alternatively, if the
amino acid Amino acids are organic compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compounds are known. The study of the properties, reactions, a ...

amino acid
residue that is changed is in a relatively unimportant part of the enzyme (e.g., a long way from the
active site
active site
), then the mutation may be selectively neutral and subject to
genetic drift Genetic drift (allelic drift or the Sewall Wright effect) is the change in the frequency of an existing gene In biology, a gene (from ''genos'' "...Wilhelm Johannsen coined the word gene to describe the Mendelian_inheritance#History, M ...

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 A chemical reaction is a process that leads to the chemical transformation of one set of chemical substance A chemical substance is a form of matter In classical physics and general chemistry, matter is any substance that has mass and t ...

chemical reaction
, 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 Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzy ...

glucokinase
, a variant of
hexokinase A hexokinase is an enzyme Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the e ...
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 cell (biology), cells as the majority of glucose entering a cell will become phosphoryla ...
. 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; it is considered to be the main Anabolism, anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and p ...

insulin
release by the
beta cell Beta cells (β cells) are a type of cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Closed spaces * Monastic cell, a small room, hut, or cave in which a monk or religious recluse liv ...
s of the
pancreas The pancreas is an organ Organ may refer to: Biology * Organ (anatomy) An organ is a group of Tissue (biology), tissues with similar functions. Plant life and animal life rely on many organs that co-exist in organ systems. A given organ ...

pancreas
, or initiation of
glycogen Glycogen is a multibranched polysaccharide Polysaccharides (), or polycarbohydrates, are the most abundant found in . They are long chain carbohydrates composed of units bound together by . This carbohydrate can react with water () usi ...

glycogen
synthesis by
liver The liver is an organ Organ may refer to: Biology * Organ (anatomy) An organ is a group of Tissue (biology), tissues with similar functions. Plant life and animal life rely on many organs that co-exist in organ systems. A given organ's t ...

liver
cells. Both these processes must only occur when glucose is abundant. 1.) The enzyme
lactate dehydrogenase Lactate dehydrogenase (LDH or LD) is an enzyme Enzymes () are s that act as s (biocatalysts). Catalysts accelerate . The molecules upon which enzymes may act are called , and the enzyme converts the substrates into different molecules kno ...
is a tetramer made of two different sub-units, the H-form and the M-form. These combine in different
combinations In mathematics Mathematics (from Ancient Greek, Greek: ) includes the study of such topics as quantity (number theory), mathematical structure, structure (algebra), space (geometry), and calculus, change (mathematical analysis, analysis). It ...
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 reaction A chemical reaction is a process that leads to the chemical transformation of one set of chemical substance A chemical substance is a form of matter 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 compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compounds are known. The study of the properties, reactions, a ...

amino acid
substitutions that change the
electric charge Electric charge is the physical property of matter that causes it to experience a force when placed in an electromagnetic field. Electric charge can be ''positive'' or ''negative'' (commonly carried by protons and electrons respectively). Like c ...
of the enzyme are simple to identify by
gel electrophoresis File:Gel Electrophoresis in DNA Fingerprinting.svg, 200px, Gel electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples. 1) DNA is extracted.2) Isola ...

gel electrophoresis
, and this forms the basis for the use of isozymes as
molecular marker A molecule is an electrically Electricity is the set of physical phenomena associated with the presence and motion Image:Leaving Yongsan Station.jpg, 300px, Motion involves a change in position In physics, motion is the phenomenon ...
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 The potato is a starch Starch or amylum is a consisting of numerous units joined by s. This is produced by most green s for energy storage. Worldwide, it is the most common carbohydrate in human diets, and is contained in large amount ...

potato starch
, but
acrylamide Acrylamide (or acrylic amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a chemical compound, compound with the general formula RC(=O)NR′R″, where R, R', and R″ represent organic compound, orga ...
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 Meteorology is a branch of the (which include and ), with a major focus on . The study of meteorology dates back , though significant progress in meteorology did not begin until the 18th century. The 19th century saw mod ...
of soluble indicator
dye A dye is a color Color (American English) or colour (Commonwealth English) is the visual perception, visual perceptual Physical property, property corresponding in humans to the categories called ''blue'', ''green'', ''red'', etc. Colo ...
s such as tetrazolium salts which become insoluble when they are by cofactors such as
NAD #REDIRECT NAD NAD or Nad may refer to: Geography * Nad, County Cork, a village in Ireland * North American Datum The North American Datum (NAD) is the horizontal datum now used to define the geodetic network in North America. A datum is a fo ...
or
NADP Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle The Calvin cycle, light-independent reactions, bio syn ...
, which generated in zones of enzyme activity. This assay method requires that the enzymes are still functional after separation (
native gel electrophoresis File:Gel Electrophoresis in DNA Fingerprinting.svg, 200px, Gel Electrophoresis is a process where an electric current is applied to DNA samples creating fragments that can be used for comparison between DNA samples. 1) DNA is extracted.2) Isola ...
), 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 that deals with genetic differences within and between s, and is a part of . Studies in this branch of examine such phenomena as , , and . Population genetics was a vital ingredient in the of the . Its pri ...
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 molecule is an electrically Electricity is the set of physical phenomena associated with the presence and motion Image:Leaving Yongsan Station.jpg, 300px, Motion involves a change in position In physics, motion is the phenomenon ...
s for this purpose. Although they have now been largely superseded by more informative
DNA Deoxyribonucleic acid (; DNA) is a molecule A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically Electricity is the set of physical ...

DNA
-based approaches (such as direct
DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides Nucleotides are organic ...

DNA sequencing
,
single nucleotide polymorphism In genetics, a single-nucleotide polymorphism (SNP ; plural ) is a substitution of a single nucleotide at a specific position in the genome that is present in a sufficiently large fraction of the population (e.g. 1% or more). For example, at a s ...
s and
microsatellites A microsatellite is a tract of repetitive DNA in which certain DNA motifs (ranging in length from one to six or more base pairs) are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within an organism's genome ...
), 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 Animals (also called Metazoa) are multicellular Multicellular organisms are or ...
s.


Other major examples

*The
cytochrome P450 Cytochromes P450 (CYPs) are a Protein superfamily, superfamily of enzymes containing heme as a cofactor (biochemistry), cofactor that functions as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are ...
isozymes play important roles in
metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life Life is a characteristic that distinguishes physical entities A bubble of exhaled gas in water In common usage and classical mechanics, a phys ...

metabolism
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 which alter membrane fluidity; and ...

steroidogenesis
. *The multiple forms of
phosphodiesterase Image:CAMP.svg, cAMP A phosphodiesterase (PDE) is an enzyme that breaks a phosphodiester bond. Usually, ''phosphodiesterase'' refers to cyclic nucleotide">phosphodiester_bond.html" ;"title="enzyme that breaks a phosphodiester bond">enzyme that ...
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