Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: Identification of an essential cysteine residue

J Pollard, S Richardson, P Lasry, T Neal, M Akhtar, Nigel Peter Botting, D Gani

Research output: Other contribution

12 Citations (Scopus)

Abstract

The mechanism of the L-threo-3-methylaspartate ammonia-lyase (EC 4.3.1.2) reaction has been probed using deuterium and solvent isotope effects with three different substrates, (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid. Each substrate appears to form a covalent adduct with the enzyme through the amination of a dehydroalanine (DehydAla-173) residue. The true substrates are N-protonated and at low pH, the alkylammonium groups are deprotonated internally in a closed solvent-excluded pocket after K+ ion, an essential cofactor, has become bound to the enzyme. At high pH, the amino groups of the substrates are able to react with the dehydroalanine residue prior to K+ ion binding. This property of the system gives rise to complex kinetics at pH 9.0 or greater and causes the formation of dead-end complexes which lack Mg2+ ion, another essential cofactor. The enzyme-substrate adduct is subsequently deaminated in two elimination processes. Hydrazines act as alternative substrates in the reverse reaction direction in the presence of fumaric acid derivatives, but cause irreversible inhibition in their absence. Borohydride and cyanide are not inhibitors. N-Ethylmaleimide also irreversibly inactivates the enzyme and labels residue Cys-361. The inactivation process is enhanced in the presence of cofactor Mg2+ ions and Cys-361 appears to serve as a base for the removal of the C-3 proton from the natural substrate, (2S,3S)-3-methylaspartic acid. The dehydroalanine residue appears to be protected in the resting form of the enzyme by generation of an internal thioether cross-link. The binding of the substrate and K+ ion appear to cause a conformational change which requires hydroxide ion. This is linked to reversal of the thioether protection step and generation of the base for substrate deprotonation at C-3. The deamination reaction displays high reverse reaction commitments and independent evidence from primary deuterium isotope effect data indicates that a thiolate acts as the base for deprotonation at C-3. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.

Original languageEnglish
Volume7
Publication statusPublished - May 1999

Keywords

  • enzymes and enzymic reactions
  • enzyme inhibitors
  • kinetics
  • isotope effects
  • PHENYLALANINE AMMONIA-LYASE
  • 3-SUBSTITUTED ASPARTIC ACIDS
  • (2S,3S)-3-METHYLASPARTIC ACID
  • CHEMICAL MECHANISM
  • PROLINE RACEMASE
  • SYN-ELIMINATION
  • FUMARIC ACIDS
  • METHYLASPARTASE
  • FRACTIONATION
  • DEHYDROALANINE

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