A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase.
Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D003545:Cysteine; D006641:Histidinol / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D010069:Oxadiazoles / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000201:enzymology*
D007093:Imidazoles; D010069:Oxadiazoles; D006641:Histidinol; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine