Isozyme
Encyclopedia
Isozymes are enzyme
s 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 isozymes 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
, isozymes (or 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 isozymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.
(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 of the same gene (described as allozymes).
Isozymes 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 mutation
s 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:
, 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 cell
s 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.
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 isozymes.
Whilst 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 (such as replacing aspartic acid
with glutamic acid
) are simple to identify by gel electrophoresis
, and this forms the basis for the use of isozymes as molecular 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 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 dye
s 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 isozymes as a laboratory technique.
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 markers 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 microsatellites), they are still amongst 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
s.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters (e.g. different KM
Michaelis-Menten kinetics
In biochemistry, Michaelis–Menten kinetics is one of the simplest and best-known models of enzyme kinetics. It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rate of enzymatic reactions, by relating...
values), or different regulatory properties.
The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (for example lactate dehydrogenase
Lactate dehydrogenase
Lactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
(LDH)).
In biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
, isozymes (or isoenzymes) are isoforms (closely related variants) of enzymes. In many cases, they are coded for by homologous
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
genes that have diverged over time. Although, strictly speaking, allozymes represent enzymes from different alleles of the same gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
, and isozymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.
Introduction
Isozymes were first described by R. L. Hunter and Clement MarkertClement Markert
Clement Lawrence Markert was an American biologist credited with the discovery of isozymes...
(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 (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
(described as isozymes) and (2) enzymes that are the product of different alleles of the same gene (described as allozymes).
Isozymes are usually the result of gene duplication
Gene duplication
Gene duplication is any duplication of a region of DNA that contains a gene; it may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome.The second copy of the gene is often free from selective pressure — that is, mutations of it have no...
, but can also arise from polyploidisation
Polyploidy
Polyploid is a term used to describe cells and organisms containing more than two paired sets of chromosomes. Most eukaryotic species are diploid, meaning they have two sets of chromosomes — one set inherited from each parent. However polyploidy is found in some organisms and is especially common...
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
Pseudogene
Pseudogenes are dysfunctional relatives of known genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell...
. However, if the mutations do not immediately prevent the enzyme from functioning, but instead modify either its function, or its pattern of gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
, then the two variants may both be favoured by natural selection
Natural selection
Natural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
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
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
s or from insertion-deletion (indel
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
) events that affect the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
coding sequence of the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
. As with any other new mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
, 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 fitnessFitness (biology)Fitness is a central idea in evolutionary theory. It can be defined either with respect to a genotype or to a phenotype in a given environment...
and be removed from the population by natural selectionNatural selectionNatural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
. - Alternatively, if the amino acidAmino acidAmino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
residue that is changed is in a relatively unimportant part of the enzyme (e.g., a long way from the active siteActive siteIn biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
), then the mutation may be selectively neutralNeutral theory of molecular evolutionThe neutral theory of molecular evolution states that the vast majority of evolutionary changes at the molecular level are caused by random drift of selectively neutral mutants . The theory was introduced by Motoo Kimura in the late 1960s and early 1970s...
and subject to genetic driftGenetic driftGenetic drift or allelic drift is the change in the frequency of a gene variant in a population due to random sampling.The alleles in the offspring are a sample of those in the parents, and chance has a role in determining whether a given individual survives and reproduces...
. - In rare cases, the mutation may result in an enzyme that is more efficient, or one that can catalyse a slightly different chemical reactionChemical reactionA chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
, in which case the mutation may cause an increase in fitness, and be favoured by natural selectionNatural selectionNatural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
.
An example of an isozyme
An example of an isozyme is glucokinaseGlucokinase
Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase occurs in cells in the liver, pancreas, gut, and brain of humans and most other vertebrates...
, a variant of hexokinase
Hexokinase
A hexokinase is an enzyme that phosphorylates a six-carbon sugar, a hexose, to a hexose phosphate. In most tissues and organisms, glucose is the most important substrate of hexokinases, and glucose-6-phosphate the most important product....
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
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
release by the beta cell
Beta cell
Beta cells are a type of cell in the pancreas located in the so-called islets of Langerhans. They make up 65-80% of the cells in the islets.-Function:...
s of the pancreas
Pancreas
The pancreas is a gland organ in the digestive and endocrine system of vertebrates. It is both an endocrine gland producing several important hormones, including insulin, glucagon, and somatostatin, as well as a digestive organ, secreting pancreatic juice containing digestive enzymes that assist...
, or initiation of glycogen
Glycogen
Glycogen is a molecule that serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue...
synthesis by liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
cells. Both of these processes must only occur when glucose is abundant, or problems occur.
Distinguishing isozymes
Isozymes (and allozymes) are variants of the same enzyme. Unless they are identical in terms of their biochemical properties, for example their substratesSubstrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
and enzyme kinetics
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
, they may be distinguished by a biochemical assay. However, such differences are usually subtle (particularly between allozymes which are often neutral variants
Neutral theory of molecular evolution
The neutral theory of molecular evolution states that the vast majority of evolutionary changes at the molecular level are caused by random drift of selectively neutral mutants . The theory was introduced by Motoo Kimura in the late 1960s and early 1970s...
). This subtlety is to be expected, because two enzymes that differ significantly in their function are unlikely to have been identified as isozymes.
Whilst isozymes may be almost identical in function, they may differ in other ways. In particular, amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
substitutions that change the electric charge
Electric charge
Electric charge is a physical property of matter that causes it to experience a force when near other electrically charged matter. Electric charge comes in two types, called positive and negative. Two positively charged substances, or objects, experience a mutual repulsive force, as do two...
of the enzyme (such as replacing aspartic acid
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
with glutamic acid
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
) are simple to identify by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, and this forms the basis for the use of isozymes as molecular 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 from potato starch
Potato starch
Potato starch is starch extracted from potatoes. The cells of the root tubers of the potato plant contain starch grains . To extract the starch, the potatoes are crushed; the starch grains are released from the destroyed cells...
, but acrylamide
Acrylamide
Acrylamide is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid, soluble in water, ethanol, ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts...
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
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...
of soluble indicator dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s such as tetrazolium salts which become insoluble when they are reduced
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
by cofactors
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
such as NAD
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, abbreviated NAD, is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide.In metabolism, NAD is involved...
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.
Isozymes and allozymes as molecular markers
Population geneticsPopulation genetics
Population genetics is the study of allele frequency distribution and change under the influence of the four main evolutionary processes: natural selection, genetic drift, mutation and gene flow. It also takes into account the factors of recombination, population subdivision and population...
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 markers for this purpose. Although they have now been largely superseded by more informative DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
-based approaches (such as direct DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
, single nucleotide polymorphism
Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
s and microsatellites), they are still amongst 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
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 higher animals, it specifies which males mate with which females, under which circumstances; recognised animal mating systems include monogamy,...
s.
Other major examples
- The cytochrome P450 isozymes play important roles in metabolismMetabolismMetabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
and steroidogenesis.
External links
- Allozyme Electrophoresis Techniques - a complete guide to starch gel eletrophoresis
- Development of new isozyme specific therapeutics - Fatty Acid Dioxygenases and Eicosanoid Hormones (Estonia)