L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site.
A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.
Hsu
L C
LC
Department of Microbiology, University of Texas, Austin 78712.
Okamoto
M
M
Snell
E E
EE
D001665:Binding Sites; D001667:Binding, Competitive; D001894:Borohydrides; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000494:pharmacology
D001894:Borohydrides; D010446:Peptide Fragments; D015091:beta-Alanine; C006977:3-chloroalanine; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; C025364:sodium borohydride; D000637:Transaminases; C053430:histidinol-phosphate aminotransferase; C010627:pyridoxamine phosphate