Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitochondrial serine transhydorxymethylases

Abstract

By using cytoplasmic and mitochondrial serine transhydroxymethylase isoenzymes from rabbit liver, it was shown that both enzymes exhibited similar ratios of serine transhydroxymethylase/threonine aldolase activities. Both enzymes catalysed the removal of the pro-S hydrogen atom of glycine, which was greatly enhanced by the presence of tetrahydrofolate. The cytoplasmic as well as the mitochondrial enzyme catalysed the synthesis of serine from glycine and [3H2]formaldehyde in the absence of tetrahydrofolate. The results are consistent with our previous suggestion that a role of tetrahydrofolate in the serine transhydroxymethylase reaction is to transport formaldehyde in and out of the active site (Jordan & Akhtar, 1970). The isoenzymes, however, showed remarkable differences in their inactivation by inhibitors. The serine transhydroxymethylase as well as the threonine aldolase activities of the cytoplasmic enzyme were inactivated in a similar fashion by chloroacetaldehyde, iodoacetamide, bromopyruvate and glycidaldehyde (2,3-epoxypropionaldehyde). These inhibitors had no effect on the two activities of the mitochondrial enzyme. The rate of inactivation of the cytoplasmic enzyme by glycidaldehyde was enhanced by the presence of glycine but decreased by the presence of serine. The implications of these results to the mechanism of catalysis and the nature of the active site of the enzymes are discussed.

A comparative study of the kinetics and stereochemistry of the serine hydroxymethyltransferase- and tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine

“…In order to form L-serine from glycine the pro-2S proton of glycine must be replaced by a hydroxymethylene group. It is therefore not surprising that serine hydroxymethyltransferase has been shown to catalyse the exchange of the pro-2S proton of glycine (Jordan & Akhtar, 1970;Akhtar et al, 1975). These studies and a more recent study by Shostak & Schirch (1988) did not produce any evidence that serine hydroxymethyltransferase could catalyse the exchange of the pro-2R proton of glycine.…”

Two Forms of Serine Transhydroxymethylase, One Absent in a Thymidylate‐less Mutant in Saccharomyces cerevisiae

“…Mechanistic and stereochemical studies showed that the cytoplasmic enzyme catalyses the aldol cleavage of allothreonine and the decarboxylation of aminomalonate as well as serine transhydroxymethylation, but the mitochondrial enzyme catalyses only the serine transhydroxymethylation [5]. However, both isoenzymes purified from rabbit liver, the cytoplasmic and the mitochondrial ones, exhibited similar ratios of serine transhydroxymethylase/threonine (allothreonine) aldolase activities [3]. The rabbit liver isoenzymes showed remarkable differences in their inactivation by inhibitors such as chloroacetaldehyde or glycinaldehyde [3].…”

“…However, both isoenzymes purified from rabbit liver, the cytoplasmic and the mitochondrial ones, exhibited similar ratios of serine transhydroxymethylase/threonine (allothreonine) aldolase activities [3]. The rabbit liver isoenzymes showed remarkable differences in their inactivation by inhibitors such as chloroacetaldehyde or glycinaldehyde [3]. These last properties have not been checked yet for the two yeast enzymic forms.…”

l‐Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase

“…The basic features of this apparatus are probably still present in the current enzymes and represent the foundation of their similar catalytic properties. An aldolase is thought to break the C a -C b bond of 3-hydroxyamino acids, most probably through a retroaldol cleavage mechanism (Scheme 1), leaving a quinonoid intermediate (IIa) that, in SHMT, is then protonated from the same side of the cleaved bond (Scheme 1, reaction 2a), maintaining the configuration of the a-carbon [51,52]. The catalytic base responsible for the abstraction of a proton from the hydroxyl group of substrates and the protonation of the quinonoid (or the pro-2S proton abstraction from glycine in the reverse direction) must be located on the re face of the cofactor, opposite to the PLP-binding lysine (Lys229 in eSHMT, Lys197 in eTA and Lys235 in cAlaRac).…”