Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Enzymes: Protein Catalysts of the Cell
Regulation of Enzymatic Activity
Genetic Control of Enzyme Synthesis

The operation of all cellular regulatory mechanisms is ultimately governed by genes and their products. Within The Cell, continuous METABOLISM/31.html">Transcription of many genes takes place, although a portion of The Genome may remain untranscribed. The Factors Determining the rate of enzyme Synthesis on Ribosomes in the Cytoplasm include both The rate of transcription and the rate of mRNA degradation.

a. Repression and Induction

Some Enzymes are referred to as constitutive, meaning that they are produced regardless of the cell's environmental conditions. For instance, Bacteria synthesize the enzymes required for Glucose Catabolism under all growth conditions. Enzymes of another group, known as inducible, are often synthesized in trace amounts only. However, when Cells are grown in the presence of substances that act as substrates for these enzymes, the latter are produced in much greater quantities. Thus, when E. coli is cultivated in the presence of lactose, a series of enzymes involved in the catabolism of this disaccharide is produced. Studies have shown that the synthesis of lactose-degrading enzymes is normally repressed. The genes encoding these Proteins are "switched off" through the action of a specific protein—a repressor possessing allosteric properties. The repressor binds to a specific region of the DNA molecule and blocks the transcription of genes responsible for the Synthesis of specific enzymes (Fig. 6-15). However, an inducer (such as lactose¹), by binding to the allosteric center of the repressor, decreases the affinity of the repressor for DNA, thereby derepressing the corresponding genes.

The synthesis of many enzymes within the cell appears to be suppressed almost constantly. The appearance of specific enzymes at a given time in an Organism or in a specific differentiated tissue results from derepression triggered by the accumulation of specific metabolites or by other, yet unknown, factors. In Eukaryotic cells, the control of enzyme synthesis can be exercised at both the transcriptional and translational levels.

Repression can not only be partially relieved by an inducer, but it can also be enhanced in the presence of the end product of a metabolic pathway. In some cases, such negative feedback-type repression is similarly mediated by an allosteric alteration of the repressor protein molecule. In eukaryotes, feedback control appears to be implemented at both the transcriptional and translational levels, as illustrated in Fig. 6-15.

The level of active enzyme in a cell is determined not only by its rate of synthesis, but by other factors as well. Some enzymes are synthesized as catalytically inactive proenzymes, which are subsequently converted into the active state, usually As a result of partial proteolysis. Finally, active enzymes may undergo degradation. This degradation occurs either randomly or via "programmed" hydrolytic Cleavage. Thus, as with other cellular components, enzyme Synthesis and degradation exist in a dynamic equilibrium. The net process is commonly referred to as protein turnover [68].

A characteristic feature of regulatory mechanisms operating at the transcriptional and translational levels is that they are relatively slow: the response time ranges from hours to sometimes days.

b. Isoenzymes (Isozymes)

An important aspect of Metabolic Regulation is that many enzymes can exist in multiple forms. As a rule, these isoenzymes are not isomers. Rather, they are similar yet chemically distinct protein molecules¹⁾. Isoenzymes have been known for a long time, but they gained special prominence several years ago when it was established that Lactate dehydrogenase in humans and most animals exists in five forms that are easily separated by Electrophoresis. Each form is a tetramer composed of subunits of two types. The tetramer with the highest electrophoretic mobility is formed by four identical type 1 subunits (also referred to as a- or H-subunits) and is often designated as a₄. The tetramer with the lowest electrophoretic mobility, ß₄, consists of four type 2 subunits (ß- or M-subunits). The other three forms (a₃ß, a₂ß₂, and aß₃) contain both types of subunits in various ratios. The synthesis of these two subunits is directed by different genes, whose activities vary among different Tissues. For example, type 1 subunits are predominantly produced in The Heart Muscle and Liver, whereas type 2 subunits are synthesized primarily in Skeletal Muscle.

¹⁾ In fact, the actual inducer is a lactose isomer, allolactose (Ch. 15, Sec. B, 1).

Why do cells produce isoenzymes? First of all, enzymes with differing kinetic properties are required to perform Functions that vary over time or depending on environmental conditions [69]. For instance, Substrate Concentration can vary widely from tissue to tissue; similar differences exist between the Mitochondria, Nucleus, and cytoplasm of a cell, as well as at various stages of organism development. In the case of lactate dehydrogenase, isoenzyme 1 is inhibited by an excess of Pyruvate—the product of The oxidation of lactate catalyzed by this enzyme:

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Although the exact nature of this product inhibition remains unclear²⁾, its physiological significance can seemingly be understood (at least to some extent) for an "aerobic" organ like the liver, where pyruvate is removed by oxidation; excessive lactate dehydrogenase activity is thereby suppressed when pyruvate accumulates. At the same time, the skeletal muscle isoenzyme 2 is not inhibited by excess pyruvate and meets the demands placed on an enzyme that must reduce pyruvate to lactate during bursts of increased muscular activity³⁾.

A more striking example of an enzyme that can exist in numerous forms is hexokinase [scheme (6-91)] [70]. Brain hexokinase is characterized by a low Michaelis constant for glucose (K_M = 0.05 mM). Consequently, it is capable of phosphorylating glucose to sustain further metabolism of this substrate even when the glucose concentration in the brain drops to very low levels. Conversely, glucokinase, the liver isoenzyme responsible for "clearing" glucose from the Blood, has a much higher K_M value (~10 mM), and its activity becomes significant only at high glucose concentrations (in blood, it is normally ~5.5 mM).

¹⁾ It is recommended to restrict the term "isoenzymes" (isozymes) solely to those Multiple Forms of enzymes whose occurrence stems from genetically determined differences in Primary Structure; it should not include forms generated by the Modification of the same Amino Acid Sequence [see Recommendations of the Commission on Biochemical Nomenclature of the IUPAC-IUB, published in J. Biol. Chem. (1977), 252, 5939–5941]. — Trans.

²⁾ The Chemical Nature of this inhibition is discussed in Ch. 8, Sec. 3.7.a [see also Sugrobova N. P., Kurganov B. I., Yakovlev V. A. (1975), 40, 281–289; Burgner J. W., Ainslie G. R., Jr., Cleland W. W., Ray W. J., Jr. (1978). Biochemistry, 17, 1646–1653; Burgner J. W., Ray W. J., Jr. (1978). Biochemistry, 17, 1654–1661]. — Trans.

³⁾ For further details, see the review by Everse J., Kaplan N. O. (1973). Adv. Enzymology, 37, 115–124. — Trans.

In addition to the causes of isoenzyme forms already discussed, one should mention proenzyme cleavage leading to The formation of multiple forms, partial Hydrolysis of enzymes, and reversible Modification of protein molecules (the latter is discussed in Sec. E, 4).

The presence of isoenzymes can also result from genetic variations in heterozygotes. Thus, if a genetically determined variant of a specific protein carries one more or one fewer positive or negative charge than the "standard" enzyme, electrophoresis of the corresponding protein fraction in heterozygotes will reveal a new isoenzyme. It should be noted that the electrophoretic method frequently used to detect isoenzymes fails to detect genetic variants in which Amino Acid Substitutions do not alter the net molecular charge.

Isoenzymes are designated in a variety of ways, but current practice assigns them numbers in order of decreasing electrophoretic mobility. Electrophoresis is usually performed at pH 7–9. Most enzymes within this pH range carry a net negative charge. The enzyme moving most rapidly toward the anode is assigned number 1. A similar convention has long been used in the electrophoresis of blood proteins. For example, globulins are numbered in order of decreasing mobility (a₁, a₂, etc.; see Supplement 2-A) [71].