The rate of synthesis of amino acids depends mainly on the amounts of the biosynthetic enzymes and on their activities. We now consider the control of enzymatic activity. The regulation of enzyme synthesis will be discussed in Chapter 31.

In a biosynthetic pathway, the first irreversible reaction, called the committed step, is usually an important regulatory site. The final product of the pathway (Z) often inhibits the enzyme that catalyzes the committed step (A → B).

This kind of control is essential for the conservation of building blocks and metabolic energy. Consider the biosynthesis of serine. The committed step in this pathway is the oxidation of 3-phosphoglycerate, catalyzed by the enzyme 3-phosphoglycerate dehydrogenase. The E. coli enzyme is a tetramer of four identical subunits, each comprising a catalytic domain and a serine-binding regulatory domain (Figure 24.17). The binding of serine to a regulatory site reduces the value of V_max for the enzyme; an enzyme bound to four molecules of serine is essentially inactive. Thus, if serine is abundant in the cell, the enzyme activity is inhibited, and so 3-phosphoglycerate, a key building block that can be used for other processes, is not wasted.

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The regulation of branched pathways is more complicated because the concentration of two products must be accounted for. In fact, several intricate feedback mechanisms have been found in branched biosynthetic pathways.

Feedback inhibition and activation. Two pathways with a common initial step may each be inhibited by its own product and activated by the product of the other pathway. Consider, for example, the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine. A common intermediate, hydroxyethyl thiamine pyrophosphate (hydroxyethyl-TPP; Section 17.1), initiates the pathways leading to all three of these amino acids. Hydroxyethyl-TPP reacts with α-ketobutyrate in the initial step for the synthesis of isoleucine. Alternatively, hydroxyethyl-TPP reacts with pyruvate in the committed step for the pathways leading to valine and leucine. Thus, the relative concentrations of α-ketobutyrate and pyruvate determine how much isoleucine is produced compared with valine and leucine. Threonine deaminase, the PLP enzyme that catalyzes the formation of α-ketobutyrate, is allosterically inhibited by isoleucine (Figure 24.18). This enzyme is also allosterically activated by valine. Thus, this enzyme is inhibited by the end product of the pathway that it initiates and is activated by the end product of a competitive pathway. This mechanism balances the amounts of different amino acids that are synthesized.

The regulatory domain in threonine deaminase is very similar in structure to the regulatory domain in 3-phosphoglycerate dehydrogenase (Figure 24.19). In the latter enzyme, regulatory domains of two subunits interact to form a dimeric serine-binding regulatory unit, and so the tetrameric enzyme contains two such regulatory units. Each unit is capable of binding two serine molecules. In threonine deaminase, the two regulatory domains are fused into a single unit with two differentiated amino acid-binding sites, one for isoleucine and the other for valine. Sequence analysis shows that similar regulatory domains are present in other amino acid biosynthetic enzymes. The similarities suggest that feedback-inhibition processes may have evolved by the linkage of specific regulatory domains to the catalytic domains of biosynthetic enzymes.

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Enzyme multiplicity. The committed step can be catalyzed by two or more isozymes, enzymes with essentially identical catalytic mechanisms, but different regulatory properties (Section 10.2). For example, the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three distinct aspartokinases, which evolved by gene duplication, catalyze this reaction in E. coli (Figure 24.20). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the mechanisms of catalysis are the same, their activities are regulated differently: one enzyme is not subject to feedback inhibition, another is inhibited by threonine, and the third is inhibited by lysine.

Cumulative feedback inhibition. A common step is partly inhibited by each of the final products, acting independently. The regulation of glutamine synthetase in E. coli is a striking example of cumulative feedback inhibition. Recall that glutamine is synthesized from glutamate, NH₄⁺, and ATP. Glutamine synthetase consists of 12 identical 50-kDa subunits arranged in two hexagonal rings that face each other. This enzyme regulates the flow of nitrogen and hence plays a key role in controlling bacterial metabolism. The amide group of glutamine is a source of nitrogen in the biosyntheses of a variety of compounds, such as tryptophan, histidine, carbamoyl phosphate, glucosamine 6-phosphate, cytidine triphosphate, and adenosine monophosphate. Glutamine synthetase is cumulatively inhibited by each of these final products of glutamine metabolism, as well as by alanine and glycine. In cumulative inhibition, each inhibitor can reduce the activity of the enzyme, even when other inhibitors are bound at saturating levels. The enzymatic activity of glutamine synthetase is switched off almost completely when all final products are bound to the enzyme.

The activity of glutamine synthetase is also controlled by reversible covalent modification—the attachment of an AMP unit by a phosphodiester linkage to the hydroxyl group of a specific tyrosine residue in each subunit. This adenylylated enzyme is less active and more susceptible to cumulative feedback inhibition than is the deadenylylated form. The covalently attached AMP unit is removed from the adenylylated enzyme by phosphorolysis.

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The adenylylation and phosphorolysis reactions are catalyzed by the same enzyme, adenylyl transferase. The adenylyl transferase is composed of two homologous halves, one half catalyzing the adenylylation reaction and the other half the phosphorolytic deadenylylation reaction. What determines whether an AMP unit is added or removed? The specificity of adenylyl transferase is controlled by a regulatory protein P_II, a trimeric protein that can exist in two forms, unmodified (P_II) or covalently bound to UMP (P_II-UMP). The complex of P_II and adenylyl transferase catalyzes the attachment of an AMP unit to glutamine synthetase, which reduces its activity. Conversely, the complex of P_II-UMP and adenylyl transferase removes AMP from the adenylylated enzyme (Figure 24.21).

This scheme of regulation immediately raises the question, how is the modification of P_II controlled? P_II is converted into P_II- UMP by the attachment of uridine monophosphate to a specific tyrosine residue (Figure 24.21). This reaction, which is catalyzed by uridylyl transferase, is stimulated by ATP and α-ketoglutarate, whereas it is inhibited by glutamine. In turn, the UMP units on P_II are removed by hydrolysis, a reaction promoted by glutamine and inhibited by α-ketoglutarate. These opposing catalytic activities are present on a single polypeptide chain and are controlled so that the enzyme does not simultaneously catalyze uridylylation and hydrolysis. In essence, if glutamine is present, the covalent modification system favors adenylylation and inactivation of glutamine synthetase.

If glutamine is absent, as indicated by the presence of its precursors α-ketoglutarate and ATP, the control system results in the deadenylylation and activation of the synthetase. The integration of nitrogen metabolism in a cell requires that a large number of input signals be detected and processed. In addition, the regulatory protein P_II also participates in regulating the transcription of genes for glutamine synthetase and other enzymes taking part in nitrogen metabolism. The evolution of covalent regulation superimposed on feedback inhibition provided many more regulatory sites and made possible a finer tuning of the flow of nitrogen in the cell. We have previously seen such a dual regulatory format in the regulation of glycogen metabolism (Section 21.5).

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