Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies.
Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism*; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001881:Borates / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D001881:Borates; D009243:NAD; D015091:beta-Alanine; C028466:ammonium borate; C006977:3-chloroalanine; C007742:3-fluoroalanine; D012694:Serine; D011732:Pyridoxal Phosphate; D014357:Trypsin; D000591:Amino Acid Isomerases; C043943:O-acetylserine; D000409:Alanine