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Regulation of metabolism

The flux of nutrients along eachmetabolic pathway is governed chiefly bytwo factors: (1) the availability of substrates on which pacemaker, or key,enzymes of the pathway can act and (2) the intracellular levels of specificmetabolites that affect the reaction rates of pacemaker enzymes. Keyenzymes are usually complex proteins that, in addition to the site at whichthe catalytic process occurs (i.e., the active site), contain sites to whichthe regulatory metabolites bind. Interactions with the appropriatemolecules at these regulatory sites cause changes in the shape of theenzyme molecule. Such changesmay either facilitate or hinder thechanges that occur at the active site. The rate of the enzymatic reaction isthus speededup or slowed down by the presence of a regulatorymetabolite.

In many cases, the specific small molecules that bind to the regulatorysites have no obvious structural similarity to the substrates of theenzymes; these small molecules are therefore termed allosteric effectors,and the regulatory sitesare termed allosteric sites. Allosteric effectors maybe formed by enzyme-catalyzed reactions in the same pathway inwhichthe enzyme regulated by the effectors functions. In this case a risein the level of the allosteric effector would affect the fluxof nutrients alongthat pathway in a manner analogous to thefeedback phenomena of

homeostatic processes. Such effectors may also be formed by enzymaticreactions in apparently unrelated pathways. In this instance the rate atwhich one metabolic pathway operates would be profoundly affected bythe rate of nutrient flux along another. It is this situation that, to a largeextent, governs the sensitive and immediately responsive coordination ofthe many metabolic routes in the cell.

End-product inhibition

A biosynthetic pathway is usually controlled by an allosteric effector

produced as the end product of that pathway, and the pacemaker enzymeon which the effector acts usually catalyzes the first step that uniquelyleads to the end product. This phenomenon, called end-product inhibition,is illustrated by the multienzyme, branched pathway for the formationfrom oxaloacetate of the aspartate family of amino acids (Figure 10).The system of interlocking controls is described in greater detail in Figure12. Asmentioned previously in this article, only plants and microorganismscan synthesize many of these amino acids, most animals requiring suchamino acids to be supplied preformed in their diets.

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Figure 12 shows that there are a number of pacemaker enzymes in thebiosynthetic route for the aspartate family of amino acids, most of whichare uniquely involved in the formation of one product. Each of theenzymes functions after a branch point in the pathway, and all areinhibited specifically by the end product that emerges from the branch

point. It is not difficult to visualize from Figure 12 how the supplies oflysine, methionine, and isoleucine required by a cell can be independentlyregulated. Threonine, however, is both an amino acid essential for proteinsynthesis and a precursor of isoleucine. If the rate of synthesis ofthreonine from aspartate were regulated as are the rates of lysine,methionine, and isoleucine, an imbalance in the supply of isoleucine mightresult. This risk is overcome in E. coli by the existence of three differentaspartokinase enzymes, all of which catalyze the first step common to theproduction of all the products derived from aspartate. Each has a differentregulatory effector molecule. Thus, one type of aspartokinaseis inhibitedby lysine, a second by threonine. The third kinase is not inhibitedby anynaturally occurring amino acid, but its rate of synthesis (see below) iscontrolled by the concentration of methionine within the cell. The triplecontrol mechanism resulting from the three different aspartokinasesensures that the accumulation of one amino acid does not shut off thesupply of aspartyl phosphate necessary for the synthesis of the others.

Another example of control through end-product inhibition also illustratesthe manner in which the operation of two biosynthetic pathways may becoordinated. Both DNA and the various types of RNA are assembled frompurine and pyrimidine nucleotides (see above The synthesis of

macromolecules: Nucleic acids and proteins); these in turn are built upfrom intermediates of central metabolic pathways(see above Thesynthesis of building blocks: Mononucleotides). The first step in thesynthesis of pyrimidine nucleotides is that catalyzed by aspartatecarbamoyltransferase [70a]. This step initiates a sequence of reactionsthat leads to the formation of pyrimidine nucleotides such as UTP and CTP[74]. Studies of aspartate carbamoyltransferase have revealed that theaffinity of this enzyme for its substrate (aspartate) is markedly decreasedby the presence of CTP. This effect can be overcome by the addition ofATP, a purine nucleotide. The enzyme can be dissociated into twosubunits: one contains the enzymatic activity and (in the dissociated form)

does not bind CTP; the other binds CTP but has no catalytic activity. Apartfrom providing physical evidence that pacemaker enzymes containdistinct catalytic and regulatory sites, the interactionof aspartatecarbamoyltransferase with the different nucleotides provides anexplanation for the control of the supply of nucleic acid precursors. If acell contains sufficient pyrimidine nucleotides (e.g., UTP), aspartatecarbamoyltransferase, the first enzyme of pyrimidine biosynthesis, isinhibited. If, however, the cell contains high levels of purine nucleotides(e.g., ATP), as required for the formation of nucleic acids, the inhibition ofaspartate carbamoyltransferase is relieved, and pyrimidines are formed.

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Positive modulation

Not all pacemaker enzymes are controlledby inhibition of their activity.Instead, some are subject to positive modulationi.e., the effector isrequired for the efficient functioning of the enzyme. Such enzymes exhibit

little activity in the absence of the appropriate allosteric effector. Oneinstance of positivemodulation is the anaplerotic fixation of carbon dioxideonto pyruvate and phosphoenolpyruvate (PEP); this examplealsoillustrates how a metabolic product ofone route controls the rate ofnutrient flow of another (see Figure 9).

The carboxylation of pyruvate in higher organisms [50] and thecarboxylation of phosphoenolpyruvate in gut bacteria [50a] occurs at asignificant rate only if acetyl coenzyme A is present. Acetyl coenzyme Aacts as a positive allosteric effector and is not broken down in the courseof the reaction. Moreover, some pyruvate carboxylases [50] and the PEPcarboxylase of gut bacteria are inhibited by four-carbon compounds (e.g.,aspartate). These substances inhibit because they interfere with thebinding of the positive effector, acetyl coenzyme A. Such enzymaticcontrols are reasonable in a physiological sense: it will be recalled thatanaplerotic formation of oxaloacetate from pyruvate or PEP is required toprovide the acceptor for the entry of acetyl coenzyme A into the TCAcycle. The reaction need occur only if acetyl coenzyme A is present insufficient amounts. On the other hand, an abundance of four-carbonintermediates obviates the necessity for forming more throughcarboxylation reactions such as [50] and [50a].

Similar reasoning, though in the opposite sense, can be applied to thecontrol of another anaplerotic sequence, the glyoxylate cycle (Figure 8).The biosynthesis of cell materials from the two-carbon compound acetateis, in principle, akin to biosynthesis from TCA cycle intermediates. In bothprocesses, it is the availability of intermediates such as PEP and pyruvatethat determines the rate at which a cell forms the many componentsproduced through gluconeogenesis. Although in the strictest sense theglyoxylate cycle has nodefined end product, PEP and pyruvate are, forthese physiological reasons, best fitted to regulate the rate at which the

glyoxylate cycle is required to operate. It is thus not unexpected that thepacemaker enzyme of the glyoxylate cycle, isocitrate lyase (reaction[52]), is allosterically inhibitedby PEP and by pyruvate.

Energy state of the cell

It is characteristic of catabolic routes that they do not lead to uniquelyidentifiable end products. The major products of glycolysis and the TCAcycle, for example, are carbon dioxide and water. Within the cell, the

concentrations of both are unlikely to vary sufficiently to allow them toserve as effective regulatory metabolites. The processes by which water is

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