Biochemistry of Amino Acids - A. Majster 1961

Intermediary Metabolism of Amino Acids
Isoleucine, Leucine, and Valine
Biosynthesis

For convenience, the transformations of these three branched-chain Amino Acids can be discussed together. The initial step in The breakdown of these Amino Acids and the final step in their Biosynthesis are Transamination reactions (see p. 210). The Structure/133.html">Discussion below focuses primarily on the METABOLISM of their carbon skeletons.

Bonner and co-workers [384] isolated a Neurospora crassa mutant requiring isoleucine and valine for growth, which served as a starting point for a series of investigations into The biosynthesis of these amino acids. Through the application of isotopic, enzymatic, and Genetic Methods, we now have a fairly comprehensive understanding of the main steps in the biosynthesis of valine, isoleucine, and leucine.

Early studies on Neurospora and Escherichia coli mutants demonstrated that the immediate precursors of valine and isoleucine are a-ketoisovaleric acid and a-keto-β-methylvaleric acid, respectively [354, 385–387]. Mutants with an absolute requirement for isoleucine and a partial requirement for valine were subsequently obtained. Such organisms fail to grow upon The addition of the corresponding a-keto acids; moreover, they accumulate a-keto analogs of valine and isoleucine when cultured on a medium containing minimal amounts of these Two amino acids [387, 388]. Based on growth studies with these mutants, it can be concluded that they have a defective transamination step in isoleucine biosynthesis. Enzymatic activity assays confirmed that these mutants lack the transaminase that catalyzes The formation of isoleucine from a-keto-β-methylvaleric acid [390]. The partial requirement for valine in these mutants is also attributable to the absence of this enzyme. Both mutant and wild-type Cells can synthesize a certain amount of valine via transamination between a-ketoisovaleric acid and Alanine or a-aminobutyric acid; however, The activity of the transaminase involved in this reaction is insufficient to supply the mutant with the valine required for optimal growth [390, 391]. Upon repeated subculturing of the mutant on a low-valine medium, an increase in valine-alanine transaminase activity is observed. Such cultures require little or no supplemental valine for optimal growth [391]. Following the discovery of these enzyme systems, it was found that alanine and a-aminobutyric acid can replace valine in the diet of the originally used mutants; these findings are consistent with the scheme proposed by several authors [390, 391]. Other Features of the transaminase systems involved in these transformations are discussed in Chapter III (p. 210).

The likely precursors of a-keto-β-methylvaleric and a-ketoisovaleric acids are a,β-dihydroxy acids, which accumulate in the cultures of certain mutants blocked at a biosynthetic step prior to the transamination reaction [392–394]. Such mutants can grow upon the addition of the corresponding dihydroxy acids; the enzyme that dehydrates these dihydroxy acids to form a-keto acids has been isolated from both E. coli and N. crassa. It has now been demonstrated that the formation of both a-ketoisovaleric and a-keto-β-methylvaleric acids is catalyzed by the same enzyme [395, 396].

A number of studies have pointed to metabolic links between Threonine, a-aminobutyric acid, and their corresponding a-keto acids with isoleucine and valine. For instance, isoleucine-deficient mutants of E. coli can utilize L- and D-threonine, homoserine, or a-aminobutyric acid for growth [386, 397]. It was shown that under these conditions, the carbon of threonine indeed serves as the source of four carbon atoms in the isoleucine molecule [396, 398] (p. 335).

Isotopic studies conducted on Neurospora and Torula utilis have yielded important new data regarding the Water/144.html">Origin of the dihydroxy acids that act as precursors for a-ketoisovaleric and a-keto-β-methylvaleric acids. Experiments with C¹⁴-labeled acetic acid suggested that the straight four-carbon chains in the valine and isoleucine molecules share a common origin [399]. However, subsequent investigations carried out in two independent laboratories demonstrated that the carbon skeletons of valine and isoleucine have different origins, despite the similarity of the reactions involved in their formation. Strassman and co-workers [400, 401] studied the incorporation of lactic acid carbon into valine in T. utilis. They found that the carboxyl carbon of lactic acid is incorporated exclusively into the carboxyl group of valine, whereas the a-carbon atom of lactic acid serves as the precursor for the C-2 and C-3 atoms of valine; the carbon of the valine methyl groups originates from the a-carbon atom of lactic acid. Of particular significance is the observation that both the C-2 and C-3 atoms of the valine molecule originate from the a-carbon atom of lactic acid, which indicates the formation of a bond between the a-carbon atoms of two lactic acid molecules during valine biosynthesis. The potential mechanism of this transformation is illustrated by the following scheme:

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According to this scheme, pyruvic acid condenses with acetaldehyde to yield a-acetolactic acid, in which a pinacol-type rearrangement causes a methyl group to shift from the a-position to the ß-position. Through a similar rearrangement, a-acetolactic acid can also give rise to a-keto-β-hydroxyisovaleric acid, which may likewise serve as a precursor of valine:

Edelberg [396], drawing on research data regarding valine biosynthesis in Neurospora, proposed a similar intramolecular rearrangement; his scheme differs slightly from the one presented above:

This reaction sequence accounts for the Formation of the dihydroxy acid as a precursor of valine. Both schemes are in agreement with experimental data on the formation of the valine carbon Skeleton and pyruvic acid from the carbon atoms of acetic acid and CO₂ [402–404].

Evidence indicates that isoleucine synthesis proceeds via an analogous pathway. It is hypothesized that acetaldehyde, derived from pyruvic acid, condenses with a-ketobutyric acid. Subsequent transformations parallel the reactions described above for valine [398, 403, 405, 406]. Alternatively, pyruvic acid and a-ketobutyric acid may initially condense to form a seven-carbon intermediate, which undergoes decarboxylation after a side-chain migration. A similar Condensation of two pyruvic acid molecules is also possible in valine biosynthesis. While data from experiments with labeled precursors are consistent with the aforementioned hypotheses, elucidating the exact Nature of the intermediates and their conversions remains a task for future research. Strassman and Weinhouse [407] calculated the theoretical distribution of carbon atoms from the methyl and carboxyl groups of acetic acid in the synthesized isoleucine molecule, based on the assumption that a-ketobutyric acid is derived from aspartic acid, which in turn is formed from oxaloacetate via The Citric Acid Cycle. The observed labeling pattern in the isolated isoleucine is in good agreement with the calculated values. The methyl- and ethyl-group Migrations envisioned by the schemes above represent a novel type of reaction, at least for biological systems, and demonstrating such transformations using enzyme preparations would be of considerable interest. Literature reports point to the possibility of analogous alkyl-group migrations in other biological systems, such as during squalene cyclization [401, 408, 409, 412].

The Role of threonine as a precursor of isoleucine is supported by experiments showing that threonine carbon is incorporated into microbially synthesized isoleucine ([396], see also [237]). Threonine serves as the source of carbon atoms 1, 2, 5, and 6 of isoleucine, with carbon atoms 1 and 2 of threonine directly transitioning into the corresponding carbon atoms of the isoleucine molecule:

These findings can be explained by the potential Cleavage of threonine into two-carbon fragments coupled with the aforementioned intramolecular rearrangement process. Threonine appears to be an obligatory precursor of isoleucine in E. coli [1100]. It has been found that certain mutants requiring isoleucine for growth lack L-Threonine dehydratase [1101] and are consequently unable to convert threonine into the a-ketobutyric acid required for isoleucine formation.

Leucine synthesis has been studied in T. utilis using labeled acetic and lactic acids [410]. The obtained data indicate that leucine is synthesized from acetic acid and the isobutyryl moiety of valine (see also [411]). Results from a study of competitive relationships among leucine precursors in E. coli using isotopic tracers [117] suggested that a-ketoisovaleric acid can serve as a precursor for leucine. Strassman and co-workers [410] proposed that a condensation occurs between a-ketoisovaleric acid and the methyl carbon of acetyl-CoA to yield a-hydroxy-a-isopropylsuccinic acid, which is subsequently converted into a-ketoisocaproic acid. This transformation is analogous to The conversion of citric acid into a-ketoglutaric acid. Leucine is then formed from a-ketoisocaproic acid via transamination: