Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.
The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace 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 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.
Teng
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Department of Biochemistry, Fels Institute for Cancer and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Grubmeyer
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D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002413:Cations, Divalent; D017124:Conserved Sequence / Q000235:genetics*; D003903:Deuterium; D006639:Histidine / Q000235:genetics* / Q000502:physiology; D006641:Histidinol / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D020005:Propanols / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
C099267:2-chloro-3-(5-imidazolyl)propanol; D002413:Cations, Divalent; D007093:Imidazoles; D008024:Ligands; D020005:Propanols; D011994:Recombinant Proteins; D006639:Histidine; D006641:Histidinol; D001216:Asparagine; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D015032:Zinc