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.
The mechanisms of the elimination of ammonia from (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-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 (2S,3S)-3-methylaspartic acid and aspartic acid are 1.0246 +/- 0.0013 and 1.0390 +/- 0.0031, respectively. The natural substrate, (2S,3S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C(beta)-H and C(alpha)-N bonds are cleaved in the same transition state. (2S)-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. (2S,3R)-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.
School of Chemistry and Centre for Biomolecular Sciences, The University of St. Andrews, Scotland, UK.
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D
Gani
C H
CH
Archer
N P
NP
Botting
J R
JR
Pollard
D000642:Ammonia-Lyases / Q000737:chemistry*; D003013:Clostridium / Q000201:enzymology; D003903:Deuterium / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D013237:Stereoisomerism
D009587:Nitrogen Isotopes; D003903:Deuterium; D000642:Ammonia-Lyases; C034881:methylaspartate ammonia-lyase