Identification of a lysine residue in the NADH-binding site of salicylate hydroxylase from Pseudomonas putida S-1.
Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
Suzuki
K
K
Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa.
Mizuguchi
M
M
Gomi
T
T
Itagaki
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin; D008239:Lysine / Q000032:analysis*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry*; D016958:Pseudomonas putida / Q000201:enzymology*; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid
D009243:NAD; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D002918:Chymotrypsin; D008239:Lysine