Stereochemical and mechanistic studies of amino acid metabolising enzymes

Akhtar, Mahmoud (1988) Stereochemical and mechanistic studies of amino acid metabolising enzymes. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The stereochemical and mechanistic features of the reactions catalysed by two pyridoxal-5'-phosphate dependent enzymes, aspartate aminotransferase (AAT) and methionine decarboxylase, and by /9-methylaspartase have been examined.

Chemical and enzymic approaches to the synthesis of 3- halogcnoaspartic acids, potential inhibitors for mammalian cytosolic aspartate aminotransferase were investigated. A convenient one step enzymic synthesis of (2R,3S)-3-chloroaspartic acid is described. (2R,3S)-3-Chloroaspartic acid was found to act as a Kcat inhibitor for AAT and a possible mechanism for inhibition is proposed.

The stereochemical courses of the decarboxylation reaction catalysed by two methionine decarboxylase isoenzymes, one from the male fern and one from a streptomyces sp. were studied. Camphanamide derivatives of the deuteriated amine products, and the corresponding reference samples, were prepared. Both enzymes catalyse decarboxylation with retention of configuration at Cat. The chiral reference samples were synthesised from (2S)- and (2R)-[4-2Hi]- f-aminobutyric acids.

The fern decarboxylase is able to utilise L-valine as a substrate, and it was expected that replacement of each methyl group of valine by a bromine atom would yield a suicide substrate. The potential inhibitors (2R.3S)- and (2R,3R)-3- bromobutyric acid were synthesised. Both of the compounds were found to be suicide inhibitors for methionine decarboxylase.

/9-Methylaspartase, an enzyme from clostridium tetanomorphwn has been used in the enantiospecific synthesis of highly functionalised 3-alky 1 and 3-halogenoaspartic acids. The precursors, 3-substituted fumaric acids were prepared and were incubated with the enzyme in the presence of high concentrations of ammonia to give the corresponding 3-substituted amino acids in good to excellent yields. The stereochemical course of these retro-physiological reactions has been determined. For each substrate nitrogen approaches from the C-2- si-face of the fumaric acid (the spatially equivalent re-face of the halogeno substrates) to give the anti- addition product. By conduction the experiment in deuterium oxide, the 3-deuteriated products were produced with a greater than 95 atom% of deuterium at C-3. The synthesis of chirally deuteriated aspartic acids by this method gives yields 2 to 3 times higher than that reported in the literature. These compounds have been used in kinetic studies.

Together with Dr.N.P.Botting, a post-doctoral research fellow in our laboratory, the rates for the forward and reverse reaction for a range of sub-strate^roduct pairs have been determined. The primary deuterium isotope effects on V and V/K were determined for aspartic, 3-methyl and 3-ethylaspartic acid and were found to be 1.0, 1.7 and 1.2 respectively. Differential 16N-isotope effects were also observed for these substrates. The results are discussed in terms of a change in mechanism with substrate structure. (DX84433)

This record has no associated files available for download.