Synthesis of isotopically labelled substrates and their use in stereochemical and mechanistic studies of enzyme reaction

Thomas, Neil Rodney (1990) Synthesis of isotopically labelled substrates and their use in stereochemical and mechanistic studies of enzyme reaction. University of Southampton, Doctoral Thesis.

Abstract

2-Amino-2-methylmalonic acid has been synthesised and has been shown to be a substrate which is decarboxylated by serine hydroxymethyltransferase (SHMT). In order to determine the stereochemical course of this reaction (2R)- and (2S)- [1-¹³C]-2-methyl-2-aminomalonic acids were prepared. The synthesis of these compounds was achieved by employing the anion of Schollkopf's bis-lactim ethers in a Claisen-type condensation with ¹³C-carbonyl-labelled electrophiles. Only acetyl chloride gave the required regio-selectivity. The resulting ketone was oxidised using the bromoform reaction. The labelled substrates were then utilised in stereochemical studies. These revealed that decarboxylation occurred stereospecifically. During the reaction, the pro-R carboxyl group of the substrate was replaced with a proton, with retention of configuration, to give (2R)-alanine. The result was not in accord with the earlier finding, by others, that the decarboxylation fo 2-aminomalonic acid by SHMT occurs non-stereospecifically. A study of the α-hydrogen exchange kinetics of 2-aminomalonic acid, using ^1H-n.m.r. indicated that the earlier observations were misinterpreted, due to rapid substrate racemisation. It was further shown that the decarboxylation of 2-aminomalonic acid was stereospecific. The role of a histidine residue which is conserved in all pyridoxal 5'-phosphate(PLP) dependent serine hydroxymethyltransferases, as well as many L-amino acid α-decarboxylases, and other PLP dependent enzymes is discussed. The ability of a mutant form of SHMT in which this histidine residue has been substituted by an asparagine residue, to decarboxylate aminoalkylmalonates has also been determined. The structural and catalytic implications of this change on the mechanism of the reaction are discussed. [4'-²H₃]-mesaconic acid has been prepared via routes involving a Favorskii-type rearrangement, or methyl cuprate addition to dimethylacetylene dicarboxylate. This compound has been used by Dr. N.P. Botting to determine the rate and order of product debinding for the deamination reaction catalysed by 3-methylaspartase (EC4.3.1.2.). A new synthetic route to L-erythro-3-methylaspartic acid has been developed, based on Schollkopf bis-lactim ether methodology. This compound has been used to show that a single 3-methylaspartase activity is responsible for catalysing the deamination of both L-threo- and L-erythro-3-methylaspartic acids. (2S,3S)-, (2R,2R)- and meso-2,3-dicarboxyaziridines have been synthesised from their appropriate tartaric acids, via intermediate cyclic-sulphates. The compounds have been used to determine the stereochemistry of the ultimate products of the enzymic amination of bromo- and chlorofumaric acids with 3-methylaspartase, as meso- and (2S,3S)-2-3-dicarboxyaziridines respectively. The ability of the aziridines to inhibit 3-methylaspartase and fumarase (EC 4.1.2.1) has been investigated. (2S,3S)-Dicarboxyaziridine is a competitive inhibitor for both enzymes.

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