L-histidinol dehydrogenase, a Zn2+-metalloenzyme.
The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.
Grubmeyer
C
C
Department of Biology, New York University, New York 10003.
Skiadopoulos
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M
Senior
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AE
D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D002614:Chelating Agents; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008667:Metalloproteins; D010618:Phenanthrolines / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D012486:Salmonella typhimurium / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
D002614:Chelating Agents; D008667:Metalloproteins; D010618:Phenanthrolines; D008345:Manganese; D014508:Urea; D000429:Alcohol Oxidoreductases; C021358:histidinol dehydrogenase; D015032:Zinc; C025205:1,10-phenanthroline