Spinach, amino acid activating enzymes

Dihydroxyacid dehydratase of the branched-chain amino acid biosynthetic pathway catalyzes the dehydration and tautomerization of 2,3-dihydroxy-3-methyl-(butyrate and pentanoate) to 2-keto-3-methyl(butyrate and pentanoate). The enzyme isolated from spinach recently has been shown to have not a [4Fe-4S] cluster, but rather a spectroscopically unusual [2Fe-2S] cluster in its active site (68,69). The EPR spectrum of the reduced enzyme is similar to that seen for Rieske Fe-S proteins (71) with a g-average of 1.91. Upon addition of substrate the g-average of the EPR spectrum shifts to 1.96 (opposite the effect of substrate on aconitase), and then reverts back to a g-average of 1.90 when only the product is present The dramatic changes in the EPR spectra upon addition of substrate suggest, in analogy to aconitase, that the Fe-S cluster may be directly involved in catalysis. 

Dihydroxyacid dehydratase (E.C. 4.2.1.9) is a ubiquitous enzyme that is involved in the biosynthesis of the branched-chain amino acids (lie, Leu and Val) and of pantothenic acid and coenzyme A. The enzyme catalyzes the elimination of water from 2,3-dihydroxyalkanoic acids (23) to 2-hydroxy-2-alkenoic acids (24), which tautomerize to 2-ketoalkanoic acids (25). The enzyme from spinach has the highest activity towards 2,3-dihydroxy-3-methylbutanoic acid (Val precursor, Scheme 11.5-4) but also accepts other substrates1341. Thus, 2,3-dihydroxybutanoic acid, 3-cyclopropyl-2,3-dihydroxybutanoic add as well as 2,3-dihydroxy-3-methylpentanoic acid are substrates. With the latter substrate a slight preference for (2R,3S)-2,3-dihydroxy-... 

O-Acetylserine is synthesized from serine and acetyl-coenzyme A in a reaction catalyzed by serine transacetylase. Brunold and Suter (1982) reported that about 35% of the activity present in spinach leaves is associated with chloroplasts. Since no activity was associated with mitochondria or peroxisomes the remaining 65% is presumably associated with the cytosol. Brunold and Suter (1982) also reported that the enzyme is almost completely inactive in the presence of 1 mM L-cysteine, confirming the earlier obrervation of Smith and Thompson (1971). Some further details of cysteine syntha.se, mostly concerning amino acid composition and the presence of multiple forms, have been reported since the previous review in this series (Diessner and Schmidt, 1981 Ikegami et al, 1987 Murakoshi et al, 1985,1986). 

Nearly 10 other amino acid dehydrogenases in addition to the above-mentioned four enzymes have been characterized as summarized in Table 1. The presence of NAD-specific glycine dehydrogenase (EC 1.4.1.10), serine dehydrogenase (EC 1.4.1.7), and NADP-specific L-amino acid dehydrogenase (EC 1.4.1.5) have been reported, but they were not substantially characterized. Tiyptophan dehydrogenase (EC 1.4.1.19, TryDH) was shown to be present in spinach leaves and to catalyze the NAD- or NADP-dependent conversion of tryptophan into indolepyruvate [38]. The enzyme is activated by the addition of calcium... 

Many plant tissues contain hydroxypyruvate reductase (o-glycerate dehydrogenase) but it is especially active in leaves (Stafford et al., 1954 Stafford and Magaldi, 1954). Tolbert et al. (1970) purified the spinach enzyme and investigated its properties. It catalyzes reduction of hydroxypyruvate to D-glyceric acid by NADH [Eq. (3)]. Although the equilibrium is toward glyceric acid rather than hydroxypyruvate, the presence of sufficient amino donor and an appropriate transaminase would allow serine synthesis by the nonphosphorylated pathway with Eq. (3) as an intermediate step. 

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