Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes a n unusual four-electron oxidation of the amino alcohol histidinol via the h istidinaldehyde intermediate to the acid product histidine with the reducti on of two molecules of NAD. An essential base, with pK(a) about 8, is invol ved in catalysis. Here we report site-directed mutagenesis studies to repla ce each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7 000- and 500-fold decreases in k(cat), respectively, with little change in K-M values. Similar loss of activity was also reported for a H261N mutant B rassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kineti c isotope effects, pH profiles, substrate rescue, and stopped-flow experime nts suggested that His-261 and His-326 are involved in proton transfers dur ing catalysis. Sensitivity to metal ion chelator and decreased affinities f or metal ions with substitutions at His-261 and His-418 suggested that thes e two residues are candidates for zinc ion ligands.