L-Histidinol dehydrogenase, a Zn 2+-metalloenzyme

Abstract

The enzymatic activity of l-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 m m MnCl 2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn 2+/mol dimer, K D = 37 μM. ZnCl 2, which prevented the MnCl 2 stimulation, also bound to the enzyme, 1.2 g-atom/ mol dimer, K D = 51 μM, and prevented Mn 2+ 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 MnCl 2 or ZnCl 2. 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 MnCl 2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl 2 or ZnCl 2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn 2+/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 Zn 2+, but which may bind Mn 2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn 2+ fulfills an essential structural and/or catalytic role.