Gas chromatography-mass spectrometry for probing the structure and mechanism of action of enzyme active sites. The role of Glu-270 in carboxypeptidase A.
A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002234:Carbodiimides; D002268:Carboxypeptidases / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005971:Glutamates / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D002234:Carbodiimides; D005971:Glutamates; D010446:Peptide Fragments; D002268:Carboxypeptidases