4 Control Mechanisms of Immediate Regulation of Metabolism

1. Substrate Concentration Effects:

Enzyme activity as mea­sured by the rate of product formation increases as a hyperbolic function as the substrate concentration is raised (Fig. 11-3) until a maximum reaction velocity (V_max) is achieved.

Excessively high substrate concen­trations may actually reduce enzyme activity.

Each enzyme, subject to experimental conditions, has a characteristic V_max as exemplified in Table 11-1 for the glycolytic enzymes in brain tissue.

If the sub­strates present in brain tissue were to occur in excess, one would expect that the reaction catalyzed by aldo­lase (which has the lowest V_max) would be the rate- limiting reaction in the sequence.

However, in vivo studies indicate that enzymes are rarely saturated by substrate. Under typical conditions, hexokinase, phosphoglucoisomerase, and aldolase operate in the pres­ence of substrate concentrations equal to or somewhat greater than the K_M value (Michaelis-Menten con­stant).

Small changes in substrate concentration do not significantly alter the rate of metabolism in this part of the glycolytic pathway. Of greater regulatory importance for these reactions is the amount of en­zyme present. The last six glycolytic enzymes in brain tissue operate at substrate levels that are significantly below the K_M value.

Therefore, small changes in sub­strate concentration would be more likely to alter the rates of their respective reactions. Under anoxic con­ditions, the rate of production of lactate is not appre­ciably increased by supplementing the tissue with glu­cose. However, supplements of compounds such as glycerol, which enter the glycolytic pathway below the level of adolase, cause an increase in lactate produc­tion.

The affinity between enzymes and their substrates affects the rates of enzyme-catalyzed reactions. For example, at branch points of metabolic pathways, two enzymes compete for the same substrate. The reac­tion catalyzed by the enzyme with the lower K_M value is favored at low substrate concentrations whereas the reaction catalyzed by the enzyme with the higher K_M value is favored at high substrate concentrations (Fig. 11-4).

Thus, when present in low concentrations, a substrate may be channeled primarily into one path­way, whereas the major direction of metabolism may shift to other pathways at higher substrate concentra­tions.

2. Effects of Salts and pH:

A variety of environ­mental factors influence the structure of proteins and as such influence the catalytic activity of enzymes. Small changes in pH or an increase (or decrease) in the salt content of the enzyme’s environment may quickly be followed by a dramatic change in the level of activity of the enzyme. A change in activity of one enzyme of a pathway will change the activity of the metabolic pathway as a whole.

3. Covalent Bond Modification:

A number of enzymes are produced in an inactive or zymogen form and must be modified through cleav­age of certain covalent bonds in. order to become ac­tive. The modification may be irreversible, as is the case with hydrolytic modification of zymogens such as pepsinogen (to form pepsin) and trypsinogen (to form trypsin).

Other enzymes may be reversibly covalently activated and deactivated. The reversible activation and deactiva­tion of glutamine synthetase is a well-studied example (Fig. 11-5). This enzyme catalyzes the conversion of glutamate to glutamine,

Glutamine synthetase is inactivated by a reversible adenylation in which 12 molecules of AMP become co­valently bonded to the hydroxyl groups of 12 tyrosine residues of the enzyme. The inactivation is catalyzed by adenylating enzyme, which uses ATP as the source of adenylate and releases pyrophosphate. Reactiva­tion is brought about through the action of deadenylating enzyme, which, in the presence of inorganic phosphate, cleaves the bonds linking the AMP mole­cules to the inactive form of the enzymes, thereby pro­ducing ADP (Fig. 11-5).

4. Isozymes:

In a number of instances, an enzyme that catalyzes a specific reaction may exist in multiple forms- in a cell or organism. These multiple forms or isozymes have different chemical structures, and can therefore be separated from one another by electrophoresis and chromatographic procedures.

Although they catalyze the same reactions, the isozymes usually have differ­ent K_M and V_max values and catalyze the reaction more or less effectively depending on the reaction condi­tions. One of the most exhaustively studied of the known isozymes is lactic dehydrogenase, which cata­lyzes one of the terminal reactions in glycolysis, that is, pyruvate + N ADH + H ⁺ <===> lactate + N AD ⁺

In rats and in a number of other vertebrates, this en­zyme is present in five forms. Each of the five forms has a molecular weight of about 134,000, consists of four polypeptide chains of about 33,500 each, and cat­alyzes the same reaction.

Each of the four polypep­tides may be of two types, usually referred to as M and H. In rat skeletal muscle tissue, the predominant form of the isozyme contains four polypeptides of the M type; in contrast in rat heart muscle, the predomi­nant form contains four polypeptides of the H type.

The other isozyme forms are made up of three H and one M, two H and two M, and one H and three M poly­peptides (usually abbreviated H₃M, H₂M₂, and HM₃). Some of the latter forms predominate in other tissues, although each tissue has some of each isozyme (Fig. 11-6).

The M₄ isozyme is prevalent in embryonic tissue and in skeletal muscle tissue. It has a low K_M and a high V_max. It is well adapted to these tissues, which are fre­quently deprived of oxygen and must depend on the energy made available during the breakdown of glu­cose to lactate. Similarly, the H₄ isozyme is well suited to the metabolism of heart muscle.

This isozyme has a high K_m and a low V_max and is inhibited by excess pyruvate. Heart muscle is primarily aerobic, convert­ing pyruvate to CO₂ and H₂O rather than to lactate. The lactic dehydrogenase enzyme is most active dur­ing emergency conditions when the oxygen supply is low.