Mechanistic studies of two amino acid racemases of broad substrate specificity from Pseudomonas striata and Aeromonas caviae.
The conversion of L-[alpha-2H]alanine in H2O and unlabeled L-alanine in 2H2O into D-alanine, under nearly irreversible conditions, with the amino acid racemase from Pseudomonas striata showed significant internal transfer of the alpha-hydrogen. This result has been interpreted as being indicative of a single base mechanism for the racemization. The relative rates of deuterium incorporation into unlabeled D- and L-methionine by the two amino acid racemases of broad substrate specificity from P. striata and Aeromonas caviae, were measured in 2H2O. The results showed a markedly different pattern, dependent upon the configuration of the initial substrate; with D-methionine as substrate deuterium is incorporated into both enantiomers at approximately the same rate, but with L-methionine as substrate deuterium is incorporated considerably faster into the D than the L enantiomer. These results argue against a single base mechanism of racemization for these enzymes and are best rationalized in terms of a double base model where only one of the bases undergoes proton (deuterium) exchange with the solvent while the amino acid is enzyme-bound. The interpretation of the earlier experiment needs to be considered in light of these results.
Department of Chemistry BG-10, University of Washington, Seattle 98195.
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D000333:Aeromonas / Q000201:enzymology*; D000409:Alanine / Q000737:chemistry; D000591:Amino Acid Isomerases / Q000032:analysis* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008968:Molecular Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D008715:Methionine; D000591:Amino Acid Isomerases; D000409:Alanine