Thus far, we have considered the conversion of N₂ into NH₄⁺ and the assimilation of NH₄⁺ into glutamate and glutamine. We turn now to the biosynthesis of the other amino acids. The pathways for the biosynthesis of amino acids are diverse. However, they have an important common feature: their carbon skeletons come from intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle. On the basis of these starting materials, amino acids can be grouped into six biosynthetic families (Figure 24.7).

The formation of asparagine from aspartate is chemically analogous to the formation of glutamine from glutamate. Both transformations are amidation reactions and both are driven by the hydrolysis of ATP. The actual reactions are different, however. In bacteria, the reaction for the asparagine synthesis is

The synthesis of glutamate by the reductive amination of α-ketoglutarate has already been discussed, as has the conversion of glutamate into glutamine. Glutamate is the precursor of two other amino acids: proline and arginine. First, the γ-carboxyl group of glutamate reacts with ATP to form an acyl phosphate. This mixed anhydride is then reduced by NADPH to an aldehyde.

Glutamic γ-semialdehyde cyclizes with a loss of H₂O in a nonenzymatic process to give Δ¹-pyrroline-5-carboxylate, which is reduced by NADPH to proline. Alternatively, the semialdehyde can be transaminated to ornithine, which is converted in several steps into arginine.

Serine is synthesized from 3-phosphoglycerate, an intermediate in glycolysis. The first step is an oxidation to 3-phosphohydroxypyruvate. This α-ketoacid is transaminated to 3-phosphoserine, which is then hydrolyzed to serine.

Serine is the precursor of glycine and cysteine. In the formation of glycine, the side-chain methylene group of serine is transferred to tetrahydrofolate, a carrier of one-carbon units that will be discussed shortly.

This interconversion is catalyzed by serine transhydroxymethylase. The side-chain methylene group of serine is then transferred to tetrahydrofolate. The conversion of serine into cysteine requires the substitution of a sulfur atom derived from methionine for the side-chain oxygen atom.

24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition

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. 

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 24.21). The regulatory domains of two subunits interact to form a dimeric serine-binding regulatory unit so that the tetrameric enzyme contains two such regulatory units. Each unit is capable of binding two serine molecules. 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.

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.

Consider, for example, the biosynthesis of the amino acids valine, leucine, and isoleucine. A common intermediate, hydroxyethyl thiamine pyrophosphate (hydroxyethyl-TPP), initiates the pathways leading to all three of these amino acids. Hydroxyethyl-TPP can react with α-ketobutyrate in the initial step for the synthesis of isoleucine. Alternatively, hydroxyethyl-TPP can react 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 enzyme that catalyzes the formation of α-ketobutyrate, is allosterically inhibited by isoleucine (Figure 24.22). This enzyme is also allosterically activated by valine. Thus, this enzyme is inhibited by the 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.

Sophisticated regulation can also evolve by duplication of the genes encoding the biosynthetic enzymes. For example, the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three distinct aspartokinases catalyze this reaction in E. coli, an example of a regulatory mechanism called enzyme multiplicity. (Figure 24.24). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the mechanisms of catalysis are essentially identical, 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.

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-kd subunits arranged in two hexagonal rings that face each other. Earl Stadtman showed that 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.