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 majority of which obtain their nitrogen from glutamate or glutamine. 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 24.5).

Most microorganisms, such as E. coli, can synthesize the entire basic set of 20 amino acids, whereas human beings cannot make 9 of them. The amino acids that must be supplied in the diet are called essential amino acids, whereas the others, which can be synthesized if dietary content is insufficient, are termed nonessential amino acids (Table 24.1). These designations refer to the needs of an organism under a particular set of conditions. For example, enough arginine is synthesized by the urea cycle to meet the needs of an adult, but perhaps not those of a growing child. A deficiency of even one amino acid results in a negative nitrogen balance. In this state, more protein is degraded than is synthesized, and so more nitrogen is excreted than is ingested.

The nonessential amino acids are synthesized by quite simple reactions, whereas the pathways for the formation of the essential amino acids are quite complex. For example, the nonessential amino acids alanine and aspartate are synthesized in a single step from pyruvate and oxaloacetate, respectively. In contrast, the pathways for the essential amino acids require from 5 to 16 steps (Figure 24.6). The sole exception to this pattern is arginine, inasmuch as the synthesis of this nonessential amino acid de novo requires 10 steps. Typically, however, it is made in only 3 steps from ornithine as part of the urea cycle. Tyrosine, classified as a nonessential amino acid because it can be synthesized in 1 step from phenylalanine, requires 10 steps to be synthesized from scratch and is essential if phenylalanine is not abundant. We begin with the biosynthesis of nonessential amino acids.

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Three α-ketoacids—α-ketoglutarate, oxaloacetate, and pyruvate—can be converted into amino acids in one step through the addition of an amino group. We have seen that α-ketoglutarate can be converted into glutamate by reductive amination (Section 23.3). The amino group from glutamate can be transferred to other α-ketoacids by transamination reactions. Thus, aspartate and alanine can be made from the addition of an amino group to oxaloacetate and pyruvate, respectively.

These reactions are carried out by pyridoxal phosphate-dependent aminotransferases. Transamination reactions are required for the synthesis of most amino acids.

In Section 23.3, we considered the mechanism of aminotransferases as applied to the metabolism of amino acids. Let us review the aminotransferase mechanism as it operates in the biosynthesis of amino acids (Figure 23.11). The reaction pathway begins with pyridoxal phosphate in a Schiff-base linkage with lysine at the aminotransferase active site, forming an internal aldimine (Figure 24.7). An amino group is transferred from glutamate to form pyridoxamine phosphate (PMP), the actual amino donor, in a multistep process. PMP then reacts with an incoming α-ketoacid to form a ketimine. Proton loss forms a quinonoid intermediate that then accepts a proton at a different site to form an external aldimine. The newly formed amino acid is released with the concomitant formation of the internal aldimine.

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Aspartate aminotransferase is the prototype of a large family of PLP-dependent enzymes. Comparisons of amino acid sequences as well as several three-dimensional structures reveal that almost all aminotransferases having roles in amino acid biosynthesis are related to aspartate aminotransferase by divergent evolution. An examination of the aligned amino acid sequences reveals that two residues are completely conserved. These residues are the lysine residue that forms the Schiff base with the PLP cofactor (lysine 258 in aspartate aminotransferase) and an arginine residue that interacts with the α-carboxylate group of the ketoacid (Figure 23.12).

An essential step in the transamination reaction is the protonation of the quinonoid intermediate to form the external aldimine. The chirality of the amino acid formed is determined by the direction from which this proton is added to the quinonoid form (Figure 24.8). The interaction between the conserved arginine residue and the α-carboxylate group helps orient the substrate so that the lysine residue transfers a proton to the bottom face of the quinonoid intermediate, generating an aldimine with an l configuration at the C_α center.

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

Thus, the products of ATP hydrolysis are AMP and PP_i rather than ADP and P_i. Aspartate is activated by adenylation rather than by phosphorylation.

We have encountered this mode of activation in fatty acid degradation and will see it again in lipid and protein synthesis.

In mammals, the nitrogen donor for asparagine is glutamine rather than ammonia as in bacteria. Ammonia is generated by hydrolysis of the side chain of glutamine and directly transferred to activated aspartate, bound in the active site. An advantage is that the cell is not directly exposed to NH₄⁺, which is toxic at high levels to human beings and other mammals. The use of glutamine hydrolysis as a mechanism for generating ammonia for use within the same enzyme is a motif common throughout biosynthetic pathways.

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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 nonessential 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 in the urea cycle (Figure 23.17).

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 cysteine and glycine. As we shall see, the conversion of serine into cysteine requires the substitution of a sulfur atom derived from methionine for the side-chain oxygen atom. 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 (Figure 24.9).

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This interconversion is catalyzed by serine hydroxymethyltransferase, a PLP enzyme that is homologous to aspartate aminotransferase. The formation of the Schiff base of serine renders the bond between its α- and β-carbon atoms susceptible to cleavage, enabling the transfer of the β-carbon to tetrahydrofolate and producing the Schiff base of glycine.

Tetrahydrofolate (also called tetrahydropteroylglutamate) is a highly versatile carrier of activated one-carbon units. This cofactor consists of three groups: a substituted pteridine, p-aminobenzoate, and a chain of one or more glutamate residues (Figure 24.9). Mammals can synthesize the pteridine ring, but they are unable to conjugate it to the other two units. They obtain tetrahydrofolate from their diets or from microorganisms in their intestinal tracts.

The one-carbon group carried by tetrahydrofolate is bonded to its N-5 or N-10 nitrogen atom (denoted as N⁵ and N¹⁰) or to both. This unit can exist in three oxidation states (Table 24.2). The most-reduced form carries a methyl group, whereas the intermediate form carries a methylene group. More-oxidized forms carry a formyl, formimino, or methenyl group. The fully oxidized one-carbon unit, CO₂, is carried by biotin rather than by tetrahydrofolate.

The one-carbon units carried by tetrahydrofolate are interconvertible (Figure 24.10). N⁵,N¹⁰-Methylenetetrahydrofolate can be reduced to N⁵-methyl tetrahydrofolate or oxidized to N⁵,N¹⁰-methenyltetrahydrofolate. N⁵,N¹⁰-Methenyltetrahydrofolate can be converted into N⁵-formiminotetrahydrofolate or N¹⁰-formyltetrahydrofolate, both of which are at the same oxidation level. N¹⁰-Formyltetrahydrofolate can also be synthesized from tetrahydrofolate, formate, and ATP. N⁵-Formyltetrahydrofolate can be reversibly isomerized to N¹⁰-formyl tetrahydrofolate or it can be converted into N⁵,N¹⁰-methenyltetrahydrofolate.

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These tetrahydrofolate derivatives serve as donors of one-carbon units in a variety of biosyntheses. Methionine is regenerated from homocysteine by transfer of the methyl group of N⁵-methyltetrahydrofolate, as will be discussed shortly. We shall see in Chapter 25 that some of the carbon atoms of purines are acquired from derivatives of N¹⁰-formyltetrahydrofolate. The methyl group of thymine, a pyrimidine, comes from N⁵, N¹⁰-methylenetetra-hydrofolate. This tetrahydrofolate derivative can also donate a one-carbon unit in an alternative synthesis of glycine that starts with CO₂ and NH₄⁺, a reaction catalyzed by glycine synthase (called the glycine cleavage enzyme when it operates in the reverse direction).

Thus, one-carbon units at each of the three oxidation levels are utilized in biosyntheses. Furthermore, tetrahydrofolate serves as an acceptor of one-carbon units in degradative reactions. The major source of one-carbon units is the facile conversion of serine into glycine by serine hydroxymethyltransferase, which yields N⁵,N¹⁰-methylenetetrahydrofolate. Serine can be derived from 3-phosphoglycerate, and so this pathway enables one-carbon units to be formed de novo from carbohydrates.

Tetrahydrofolate can carry a methyl group on its N-5 atom, but its transfer potential is not sufficiently high for most biosynthetic methylations. Rather, the activated methyl donor is usually S-adenosylmethionine (SAM), which is synthesized by the transfer of an adenosyl group from ATP to the sulfur atom of methionine.

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The methyl group of the methionine unit is activated by the positive charge on the adjacent sulfur atom, which makes the molecule much more reactive than N⁵-methyltetrahydrofolate. The synthesis of S-adenosylmethionine is unusual in that the triphosphate group of ATP is split into pyrophosphate and orthophosphate; the pyrophosphate is subsequently hydrolyzed to two molecules of P_i. S-Adenosylhomocysteine is formed when the methyl group of S-adenosylmethionine is transferred to an acceptor. S-Adenosylhomocysteine is then hydrolyzed to homocysteine and adenosine.

Methionine can be regenerated by the transfer of a methyl group to homocysteine from N⁵-methyltetrahydrofolate, a reaction catalyzed by methionine synthase (also known as homocysteine methyltransferase).

The coenzyme that mediates this transfer of a methyl group is methylcobalamin, derived from vitamin B₁₂. In fact, this reaction and the rearrangement of l-methylmalonyl CoA to succinyl CoA, catalyzed by a homologous enzyme, are the only two B₁₂-dependent reactions known to take place in mammals. Another enzyme that converts homocysteine into methionine without vitamin B₁₂ also is present in many organisms.

These reactions constitute the activated methyl cycle (Figure 24.11). Methyl groups enter the cycle in the conversion of homocysteine into methionine and are then made highly reactive by the addition of an adenosyl group, which makes the sulfur atoms positively charged and the methyl groups much more electrophilic. The high transfer potential of the S-methyl group enables it to be transferred to a wide variety of acceptors. Among the acceptors modified by S-adenosylmethionine are specific bases in bacterial DNA. For instance, the methylation of DNA protects bacterial DNA from cleavage by restriction enzymes (Section 9.3). Methylation is also important for the synthesis of phospholipids (Section 26.1).

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S-Adenosylmethionine is also the precursor of ethylene, a gaseous plant hormone that induces the ripening of fruit. S-Adenosylmethionine is cyclized to a cyclopropane derivative that is then oxidized to form ethylene. The Greek philosopher Theophrastus recognized more than 2000 years ago that sycamore figs do not ripen unless they are scraped with an iron claw. The reason is now known: Wounding triggers ethylene production, which in turn induces ripening.

In addition to being a precursor of methionine in the activated methyl cycle, homocysteine is an intermediate in the synthesis of cysteine. Serine and homocysteine condense to form cystathionine. This reaction is catalyzed by cystathionine β-synthase. Cystathionine is then deaminated and cleaved to cysteine and α-ketobutyrate by cystathionine γ-lyase or cystathionase. Both of these enzymes utilize PLP and are homologous to aspartate aminotransferase. The net reaction is

Note that the sulfur atom of cysteine is derived from homocysteine, whereas the carbon skeleton comes from serine.

People with elevated serum levels of homocysteine (homocysteinemia) or the disulfide-linked dimer homocystine (homocystinuria) have an unusually high risk for coronary heart disease and arteriosclerosis. The most common genetic cause of high homocysteine levels is a mutation within the gene encoding cystathionine β-synthase. High levels of homocysteine appear to damage cells lining blood vessels and to increase the growth of vascular smooth muscle. The amino acid raises oxidative stress as well and has also been implicated in the development of type 2 diabetes (Section 27.3). The molecular basis of homocysteine’s action has not been clearly identified, but may result from stimulation of the inflammatory response. Vitamin treatments are sometimes effective in reducing homocysteine levels in some people. Treatment with vitamins maximizes the activity of the two major metabolic pathways processing homocysteine. Pyridoxal phosphate, a vitamin B₆ derivative, is necessary for the activity of cystathionine β-synthase, which converts homocysteine into cystathione; tetrahydrofolate, as well as vitamin B₁₂, supports the methylation of homocysteine to methionine.

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We turn now to the biosynthesis of essential amino acids. These amino acids are synthesized by plants and microorganisms, and those in the human diet are ultimately derived primarily from plants. The essential amino acids are formed by much more complex routes than are the nonessential amino acids. The pathways for the synthesis of aromatic amino acids in bacteria have been selected for discussion here because they are well understood and exemplify recurring mechanistic motifs.

Phenylalanine, tyrosine, and tryptophan are synthesized by a common pathway in E. coli (Figure 24.12). The initial step is the condensation of phosphoenolpyruvate (a glycolytic intermediate) with erythrose 4-phosphate (a pentose phosphate pathway intermediate). The resulting seven-carbon open-chain sugar is oxidized, loses its phosphoryl group, and cyclizes to 3-dehydroquinate. Dehydration then yields 3-dehydroshikimate, which is reduced by NADPH to shikimate. The phosphorylation of shikimate by ATP gives shikimate 3-phosphate, which condenses with a second molecule of phosphoenolpyruvate. The resulting 5-enolpyruvyl intermediate loses its phosphoryl group, yielding chorismate, the common precursor of all three aromatic amino acids. The importance of this pathway is revealed by the effectiveness of glyphosate (commercially known as Roundup), a broad-spectrum herbicide. This compound is an uncompetitive inhibitor of the enzyme that produces 5-enolpyruvylshikimate 3-phosphate. It blocks aromatic amino acid biosynthesis in plants but is fairly nontoxic in animals because they lack the enzyme.

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The pathway bifurcates at chorismate. Let us first follow the prephenate branch (Figure 24.13). A mutase converts chorismate into prephenate, the immediate precursor of the aromatic ring of phenylalanine and tyrosine. This fascinating conversion is a rare example of an electrocyclic reaction in biochemistry, mechanistically similar to the well-known Diels–Alder reaction in organic chemistry. Dehydration and decarboxylation yield phenylpyruvate. Alternatively, prephenate can be oxidatively decarboxylated to p-hydroxyphenylpyruvate. These α-ketoacids are then transaminated to form phenylalanine and tyrosine.

The branch starting with anthranilate leads to the synthesis of tryptophan (Figure 24.14). Chorismate acquires an amino group derived from the hydrolysis of the side chain of glutamine and releases pyruvate to form anthranilate. Anthranilate then condenses with 5-phosphoribosyl-1-pyrophosphate (PRPP), an activated form of ribose phosphate. PRPP is also an important intermediate in the synthesis of histidine, pyrimidine nucleotides, and purine nucleotides (Sections 25.1 and 25.2). The C-1 atom of ribose 5-phosphate becomes bonded to the nitrogen atom of anthranilate in a reaction that is driven by the release and hydrolysis of pyrophosphate. The ribose moiety of phosphoribosylanthranilate undergoes rearrangement to yield 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. This intermediate is dehydrated and then decarboxylated to indole-3-glycerol phosphate. Tryptophan synthase completes the synthesis of tryptophan with the removal of the side chain of indole-3-glycerol phosphate, yielding glyceraldehyde 3-phosphate, and its replacement with the carbon skeleton of serine.

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The E. coli enzyme tryptophan synthase, an α₂β₂ tetramer, can be dissociated into two α subunits and a β₂ dimer (Figure 24.15). The α subunit catalyzes the formation of indole from indole-3-glycerol phosphate, whereas each β subunit has a PLP-containing active site that catalyzes the condensation of indole and serine to form tryptophan. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiff base of aminoacrylate. This reactive intermediate is attacked by indole to give tryptophan.

The synthesis of tryptophan poses a challenge. Indole, a hydrophobic molecule, readily traverses membranes and would be lost from the cell if it were allowed to diffuse away from the enzyme. This problem is solved in an ingenious way. A 25-Å-long channel connects the active site of the α subunit with that of the adjacent β subunit in the α₂β₂ tetramer (Figure 24.16). Thus, indole can diffuse from one active site to the other without being released into bulk solvent. Isotopic-labeling experiments showed that indole formed by the α subunit does not leave the enzyme when serine is present. Furthermore, the two partial reactions are coordinated. Indole is not formed by the α subunit until the highly reactive aminoacrylate is ready and waiting in the β subunit. We see here a clear-cut example of substrate channeling in catalysis by a multienzyme complex. Channeling substantially increases the catalytic rate. Furthermore, a deleterious side reaction—in this case, the potential loss of an intermediate—is prevented. We shall encounter other examples of substrate channeling in Chapter 25.

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