The 3-methylaspartase reaction probed using 2H- and 15N-Isotope effects for three substrates: a flip from a concerted to a carbocationic amino-Enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S

Abstract

The mechanisms of the elimination of ammonia from (2 S,3 S)-3-methylaspartic acid, (2 S)-aspartic acid and (2 S,3 R)-3-methylaspartic acid, catalysed by the enzyme l- threo-3-methylaspartase ammonia-lyase (EC 4.3.1.2) have been probed using 15N-isotope effects. The 15N-isotope effects for V/K for both (2 S,3 S)-3-methylaspartic acid and aspartic acid are 1.0246±0.0013 and 1.0390±0.0031, respectively. The natural substrate, (2 S,3 S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C β–H and C α–N bonds are cleaved in the same transition state. (2 S)-Aspartic acid appears to follow the same mechanistic pathway, but deprotonation of the conjugate acid of the base for C-3 is kinetically important and influences the extent of 15N-fractionation. (2 S,3 R)-3-Methylaspartic acid is deaminated via a stepwise carbocationic mechanism. Here we elaborate on the proposed model for the mechanism of methylaspartase and propose that a change in stereochemistry of the substrate induces a change in the mechanism of ammonia elimination. 5 5 Abbreviations: Bis Tris Propane, 1,3-bis [tris(Hydroxymethyl)-methylamino]propane; HPLC, high-performance liquid chromatography; NEM, N-ethylmaleimide; NMR, nuclear magnetic resonance; UV, ultra-violet; TLC, thin-layer chromatography; Tris, (Tris[hydroxymethyl]-aminomethane); GC–MS, gas chromatography–mass spectrometry; SIR-GC–MS, single ion recording-gas chromatography–mass spectrometry 1