| pubmed-article:8314784 | pubmed:abstractText | The 4-electron oxidoreductase L-histidinol dehydrogenase (HDH, EC 1.1.1.23) oxidizes the amino alcohol histidinol to histidine via an aldehyde-level intermediate at a single active site. The enzyme contains two Zn2+ per dimer, and treatment with metal chelators causes a metal-reversible inactivation. NAD-linked aldehyde oxidations, for which glyceraldehyde-3-phosphate dehydrogenase has served as the major paradigm, are thought to proceed via cysteine-based thiohemiacetals. Sequenced forms of HDH contain two conserved cysteine residues, Cys-116 and Cys-153 in the Salmonella typhimurium enzyme, and in previous work we have shown that HDH is inactivated by active site modification of Cys-116 by the reagent 4-nitro-7-chlorobenzadioxazole. Thus, Cys-116 is an excellent candidate for the active site nucleophile in HDH. In the current studies we show that treatment of HDH with the Zn2+ chelator 1,10-phenanthroline exposes Cys-116 to specific modification by iodoacetate, resulting in irreversible loss of activity. Site-specific mutagenesis was used to explore the roles of the conserved cysteine residues. The mutant enzymes C116S, C153S, C116A, and C153A and the double mutant C116,153A were each overproduced and purified to homogeneity. All mutant enzymes showed normal kcat and Km values for catalysis. The double mutant protein was unstable, and the single mutants also lose significant activities over a 3-h period during which wild-type enzyme retains full activity. The C116S mutant, and to a lesser extent the C116A mutant, were sensitive to the presence of EDTA in the assay medium, but the other mutants or wild-type enzyme were not, suggesting that Cys-116 may be near, but probably not liganded to, the bound metal ion. The results clearly indicate that HDH does not use a cysteine-based thiohemiacetal as a catalytic intermediate, requiring a new paradigm for NAD-linked aldehyde oxidation. Some models for the reaction are presented and discussed. | lld:pubmed |
