CONSERVED CYSTEINE RESIDUES OF HISTIDINOL DEHYDROGENASE ARE NOT INVOLVED IN CATALYSIS - NOVEL CHEMISTRY REQUIRED FOR ENZYMATIC ALDEHYDE OXIDATION

The 4-electron oxidoreductase L-histidinol dehydrogenase (HDH, EC 1.1. 1.23) oxidizes the amino alcohol histidinol to histidine via an aldehy de-level intermediate at a single active site. The enzyme contains two Zn2+ per dimer, and treatment with metal chelators causes a metal-rev ersible inactivation. NAD-linked aldehyde oxidations, for which glycer aldehyde-3-phosphate dehydrogenase has served as the major paradigm, a re thought to proceed via cysteine-based thiohemiacetals. Sequenced fo rms of HDH contain two conserved cysteine residues, Cys-116 and Cys-15 3 in the Salmonella typhimurium enzyme, and in previous work we have s hown that HDH is inactivated by active site modification of Cys-116 by the reagent 4-nitro-7-chlorobenzadioxazole. Thus, Cys-116 is an excel lent candidate for the active site nucleophile in HDH. In the current studies we show that treatment of HDH with the Zn2+ chelator 1,10-phen anthroline exposes Cys-116 to specific modification by iodoacetate, re sulting in irreversible loss of activity. Site-specific mutagenesis wa s used to explore the roles of the conserved cysteine residues. The mu tant enzymes C116S, C153S, C116A, and C153A and the double mutant C116 ,153A were each overproduced and purified to homogeneity. All mutant e nzymes showed normal k(cat) and K(m) values for catalysis. The double mutant protein was unstable, and the single mutants also lose signific ant activities over a 3-h period during which wild-type enzyme retains full activity. The C116S mutant, and to a lesser extent the C116A mut ant, were sensitive to the presence of EDTA in the assay medium, but t he 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 r esults clearly indicate that HDH does not use a cysteine-based thiohem iacetal as a catalytic intermediate, requiring a new paradigm for NAD- linked aldehyde oxidation. Some models for the reaction are presented and discussed.