Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Enzymes
Isoenzymes

Isoenzymes, or isozymes, are Multiple Forms of an enzyme that catalyze the same reaction but differ in their Physical and Chemical properties, notably in substrate affinity, maximum reaction velocity (activity), electrophoretic mobility, or regulatory properties.

Class="center">

Fig. 4.5. Models of The Structure of certain oligomeric Enzymes.

a - Glutamate dehydrogenase molecule, consisting of 6 protomers (total molecular weight 336,000); b - RNA polymerase molecule; c - half of a catalase molecule; d - Pyruvate dehydrogenase molecular complex.

Living organisms contain enzymes whose molecules consist of two or more subunits with identical or different primary, secondary, or tertiary structures. These subunits are frequently referred to as protomers, while the assembled oligomeric molecule is called a multimer (Fig. 4.5; see Chapter 1).

It is believed that the oligomerization process confers enhanced stability and resistance upon protein subunits against Denaturing Agents, including heat, proteinases, etc. However, current knowledge does not yet provide a definitive answer regarding the essential role of quaternary structure in the catalytic activity of enzymes, as there are still no Methods available to disrupt solely The quaternary structure under mild conditions. Conventional harsh Treatment methods (extreme pH values, high concentrations of guanidine hydrochloride, or urea) lead to the destruction not only of the quaternary structure but also of the secondary and tertiary structures of a stable oligomeric enzyme, leaving its protomers denatured and, consequently, devoid of biological activity.

It should be noted that there are no covalent, primary valence bonds between the subunits. The bonds are predominantly non-covalent, which is why such enzymes dissociate into protomers rather easily. A striking feature of these enzymes is that The activity of the entire complex depends on the precise arrangement and packing of the individual subunits. If genetically distinct subunits can exist in more than one form, then an enzyme composed of two or more types of subunits combined in varying quantitative proportions can likewise exist in several similar, yet distinct forms. Such enzyme variants are termed isoenzymes (isozymes, or, less frequently, isozymic forms). Specifically, if an enzyme consists of 4 subunits of two different types — H and M (Heart and Muscle) — the active enzyme may represent one of the following combinations: HHHH, HHHM, HHMM, HMMM, MMMM, or H₄, H₃M, H₂M₂, HM₃, M₄, corresponding to the LDH₁, LDH₂, LDH₃, LDH₄, and LDH₅ isoenzymes. Furthermore, the Synthesis of the H- and M-types is directed by different genes and is expressed differentially across various Organs.

In some cases, the subunits have an almost identical structure, and each contains a catalytically Active Site (for example, $\beta$-galactosidase, which consists of 4 subunits). In other cases, the subunits are non-identical. An example of the latter is Tryptophan synthase, which consists of 2 subunits, each possessing its own (albeit secondary) enzymatic activity; however, only when combined into the macromolecular structure do both subunits exhibit full tryptophan synthase activity.

The term "multiple forms of an enzyme" applies to Proteins catalyzing the same reaction that occur naturally within organisms of the same species. The term "isoenzyme" is strictly applicable only to those multiple forms of enzymes that arise from genetically determined differences in the Primary Cell/13.html">Protein Structure (and not to forms generated by the modification of a single primary sequence).

One of the most thoroughly investigated enzymes whose multiplicity of forms has been studied in detail using gel Electrophoresis is LDH, which catalyzes the reversible conversion of pyruvic acid into lactic acid. Five LDH isoenzymes are formed from 4 subunits of approximately equal size but of two different types. Because H-protomers carry a more pronounced negative charge at pH 7.0–9.0 than M-protomers, the isoenzyme composed of 4 H-type subunits (H₄) migrates most rapidly toward the positive electrode (anode) in an electric field. The M₄ isoenzyme moves toward the anode at the lowest speed, while the remaining isoenzymes occupy intermediate positions. It must be emphasized that while LDH isoenzymes possess nearly identical enzymatic activity, they differ in certain physicochemical properties—such as molecular weight, electrophoretic mobility, and response to activators and inhibitors. Nevertheless, each normal tissue is characterized by a specific proportion of these forms (the isoenzyme profile) of LDH. For instance, H₄ (i.e., LDH₁) predominates in heart muscle, whereas M₄ (LDH₅) predominates in skeletal Muscles and the Liver (Fig. 4.6). These circumstances are widely utilized in clinical practice, as detecting the appearance of LDH isoenzymes (along with several Other Enzymes) in Blood serum can be of great value for the Cytology/practical/136.html">Differential Diagnosis OF organic and functional disorders of organs and Tissues. Changes in the serum levels of isoenzymes allow clinicians to assess both the Topography of the pathological process and the severity of damage to the organ or tissue.

Fig. 4.6. Distribution and relative amounts of LDH isoenzymes in various organs. Extracts were applied at the baseline marked as "Start". Under the given experimental conditions (pH), four of the LDH isoenzymes migrate toward the anode, and one (LDH₅) migrates toward the cathode. The major LDH isoforms for a given organ are highlighted in red.