Fundamentals of Biochemistry - A. A. Anisimov 1986

Enzymes
Multiple molecular forms of enzymes and isozymes

Multiple molecular forms of Enzymes (MMFE) refer to a group of enzymes that perform an identical catalytic function in a single biological species, yet differ in Structure and A number of physicochemical properties. Various Electrophoresis Methods followed by the specific detection of zones with identical enzymatic activity are most commonly used to separate MMFE. On electrophoregrams, zones of MMFE activity are designated by Arabic numerals in order of decreasing anodic mobility.

The existence of MMFE is of definite biological significance. It has been demonstrated, for instance, that under changing environmental conditions, THE SPECTRUM OF MMFE in a Cell can shift, enabling the Organism to better adapt to external factors. Different molecular forms of enzymes play a crucial role in differentiation and development. Thus, shifts in The ratio of MMFE (their number, The activity of each form, and stability) serve as one of The regulatory mechanisms for metabolic processes.

The initial studies on identifying MMFE were conducted on Lactate dehydrogenase (LDH), which catalyzes the redox conversion of lactate to Pyruvate and vice versa. Electrophoretic analysis of this enzyme typically reveals five fractions (1–5) exhibiting LDH activity (Fig. 3.22). All five molecular forms have nearly identical molecular weights ($M$ 134,000) and consist of four polypeptide chains, each with a relative Molecular Weight of 33,500.

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Fig. 3.22. Multiple molecular forms of lactate dehydrogenase (L) and their Separation by electrophoresis (5)

Despite their similar molecular weights, The polypeptide chains are non-identical and can be of two types: M (from Muscle) and H (from Heart). Both types of chains contain the coenzyme NAD(H⁺), but differ in several Amino Acids, which determines their different electrophoretic mobility.

M- and H-types of polypeptide chains are encoded by different genes. Individual chains lack enzymatic activity, while The formation of a tetramer through various combinations of the two chain types yields an active enzyme. In Muscle tissue, the M₄ tetramer predominates, characterized by the lowest anodic mobility, whereas in cardiac tissue, the H₄ tetramer predominates, exhibiting the highest mobility toward the anode. The remaining three MMFE of LDH are designated as M₃H₁, M₂H₂, and M₁H₃. The M₄ and M₃H₁ forms are predominantly found in Tissues where Glycolysis serves as the energy source (skeletal Muscles, embryonic tissues), while MH₃ and H₄ are prevalent in tissues characterized by aerobic METABOLISM (heart muscle).

Currently, A large number of enzymes are known to be represented in the organism by multiple molecular forms. The causes leading to the appearance of MMFE can vary; consequently, the International Commission on Biochemical Nomenclature has developed a Classification of molecular forms of enzymes. Among MMFE, there are both genetically determined forms—conventionally termed Isoenzymes or isozymes (which differ from one another in Primary Structure)—and forms arising from epigenetic changes (at the post-translational level).

All MMFE are divided into six classes.

1. Genetically independent Proteins. These are enzymes synthesized from different genes. In Multicellular Organisms, they often feature distinct intracellular and tissue localization: for example, pyruvate kinase, enolase, and fructose-bisphosphate aldolase in muscle and Liver tissues, and malate dehydrogenase along with a number of aminotransferases in Mitochondria and the Cytosol.

2. Heteropolymers (hybrids) of two or more non-covalently linked polypeptide chains. Examples of molecular forms of enzymes in this class include LDH, Alcohol dehydrogenase, and creatine kinase.

3. Genetic variants (allelosymes). Allelosymes occur in organisms heterozygous for genes encoding a given enzyme. This class includes mutant enzyme forms. This is a very extensive class of molecular forms of enzymes, encompassing, for example, human glucose-6-phosphate dehydrogenase, adenosine deaminase, and many others.

4. Conjugated or derived proteins. This class of MMFE includes enzyme forms resulting from the covalent addition or removal of specific groups to or from a protein. Such modifications are typically accompanied by Changes in the enzymatic activity and certain PHYSICOCHEMICAL PROPERTIES OF the enzyme. Modifications may involve phosphorylation–dephosphorylation (Glycogen phosphorylase, glycogen synthase, fructose-1,6-bisphosphatase), adenylylation–deadenylylation (Glutamine Synthetase from E. coli), oxidation of sulfhydryl groups (xanthine oxidase, lipoamide dehydrogenase), glycosylation (variation in the number of carbohydrate residues has been demonstrated for bovine liver β-glucuronidase, Penicillium vitale glucose oxidase, and bovine pancreatic DNase and RNase), amidation of Asp and Glu residues (differing degrees of amidation were found in the alkaline protease from Streptomyces rectus), and proteolytic Cleavage of peptide bonds (aldolase).

5. Oligomers of a single subunit. When an enzyme possesses a quaternary structure, different molecular forms can arise from the assembly of varying numbers of identical polypeptide chains into The quaternary structure. Thus, β-glucosidase is active in the form of mono-, di-, tetra-, and octamers. Varying degrees of oligomerization as a cause of MMFE have been established for Glutamate dehydrogenase, cholinesterase, and a number of Other Enzymes.

6. Conformationally differing forms (conformers). Conformers are proteins that differ in conformation despite having the same Amino Acid Sequence. Differences in the Spatial Structure of the protein associated with the number of charged groups on the molecular surface lead to differing electrophoretic mobility. All allosterically modified enzymes also belong to this 6th class.

Thus, the term MMFE can be used as the most general designation for a group of enzymes found within a single biological species that possess identical catalytic Specificity. It should be used regardless of the cause of their appearance. The term isoenzyme or isozyme is applicable only to those MMFE whose appearance is associated with genetically determined differences in primary structure (classes 1–3), rather than those caused by other factors while maintaining the same primary structure (classes 4–6).