Mechanistic studies of the enzyme 3-methylaspartate ammonia-lyase
Mechanistic studies of the enzyme 3-methylaspartate ammonia-lyase
3-Methylaspartate ammonia-lyase (E.C. 4.3.1.2), catalyses the reversible elimination of ammonia from methylaspartic acid. The enzyme from Clostridium tetanomorphum has been purified to homogeneity in three stages using a new method; acetone fractionation of the cell extracts, Sephadex G-150 gel exclusion chromatography and TSK DEAE-5PW ion exchange chromatography. Contrary to literature reports of a heterotetrameric structure the active enzyme was found to possess a homodimeric structure, with a subunit molecular weight of 50,000. The N-terminal sequence of the subunit was determined, using the pulsed-liquid phase Edman degradation method. The protein was subjected to the Edman cycle 40 times and the first 26 amino acids were clearly identifiable. Comparison with the N-terminal sequence of the functionally related enzymes aspartase (E.C. 4.3.1.1) and fumarase (E.C. 4.2.1.2) showed that there was no homology between these enzymes and 3-methylaspartate ammonia-lyase. The enzyme was used in the enantiospecific syntheses of a range of highly functionalised 3-substituted aspartic acids, from ammonia and 2-halogeno- or 2-alkylfumaric acids. 3-Chloro and 3-bromoaspartic acids were shown to inhibit the enzyme, probably through the alkylation of a catalytically essential active site base/nucleophile. These halogenoaspartic acids also underwent intramolecular cyclisation to give aziridine-2,3-dicarboxylic acid. At pH 9.0 each gave a different diastereomer, indicating that different cyclisation mechanisms operate. Together with Dr N P Botting, the kinetic parameters, Vmax and KM, were determined for the amination of a range 2-substituted fumaric acids and for the deamination of some 3-substituted aspartic acids. The kinetic data, together with recent results from our laboratory on the deuterium and nitrogen kinetic isotope effects and double isotope fractionation studies is used to construct a mechanistic scheme for the enzyme catalysed reaction. We propose that the elimination of ammonia from 3-methylaspartic acid occurs via a concerted (E2 type) mechanism.
University of Southampton
1989
Cohen, Mark Adam
(1989)
Mechanistic studies of the enzyme 3-methylaspartate ammonia-lyase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
3-Methylaspartate ammonia-lyase (E.C. 4.3.1.2), catalyses the reversible elimination of ammonia from methylaspartic acid. The enzyme from Clostridium tetanomorphum has been purified to homogeneity in three stages using a new method; acetone fractionation of the cell extracts, Sephadex G-150 gel exclusion chromatography and TSK DEAE-5PW ion exchange chromatography. Contrary to literature reports of a heterotetrameric structure the active enzyme was found to possess a homodimeric structure, with a subunit molecular weight of 50,000. The N-terminal sequence of the subunit was determined, using the pulsed-liquid phase Edman degradation method. The protein was subjected to the Edman cycle 40 times and the first 26 amino acids were clearly identifiable. Comparison with the N-terminal sequence of the functionally related enzymes aspartase (E.C. 4.3.1.1) and fumarase (E.C. 4.2.1.2) showed that there was no homology between these enzymes and 3-methylaspartate ammonia-lyase. The enzyme was used in the enantiospecific syntheses of a range of highly functionalised 3-substituted aspartic acids, from ammonia and 2-halogeno- or 2-alkylfumaric acids. 3-Chloro and 3-bromoaspartic acids were shown to inhibit the enzyme, probably through the alkylation of a catalytically essential active site base/nucleophile. These halogenoaspartic acids also underwent intramolecular cyclisation to give aziridine-2,3-dicarboxylic acid. At pH 9.0 each gave a different diastereomer, indicating that different cyclisation mechanisms operate. Together with Dr N P Botting, the kinetic parameters, Vmax and KM, were determined for the amination of a range 2-substituted fumaric acids and for the deamination of some 3-substituted aspartic acids. The kinetic data, together with recent results from our laboratory on the deuterium and nitrogen kinetic isotope effects and double isotope fractionation studies is used to construct a mechanistic scheme for the enzyme catalysed reaction. We propose that the elimination of ammonia from 3-methylaspartic acid occurs via a concerted (E2 type) mechanism.
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Published date: 1989
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Local EPrints ID: 461219
URI: http://eprints.soton.ac.uk/id/eprint/461219
PURE UUID: 79aa1a37-5429-4592-ac64-8638be86e03a
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Date deposited: 04 Jul 2022 18:39
Last modified: 04 Jul 2022 18:39
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Author:
Mark Adam Cohen
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