Activation/attenuation model for RNase H - A one-metal mechanism with second-metal inhibition

Ribonucleases H (RNases H) comprise a family of metal-dependent enzymes tha t catalyze the hydrolysis of the 3'-O-P bond of RNA in RNA. DNA hybrids. Th e mechanism by which RNases H use active-site metal(s) for catalysis is unc lear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HN RNase H domain, two models explaining RNase H metal depende nce have been proposed: a one-metal mechanism and a two-metal mechanism, In this paper, we show that the Mn2+-dependent activity off. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the pr esence of Mn2+ is complex, with activation and inhibition of the enzyme at low and high Mn2+ concentrations, respectively. Mutations at Asp-134 result in a partial loss of this inhibition, with Little effect on activation. Ne utralization of His-124 by mutation to Ala results in an enzyme with a sign ificantly decreased specific activity and an absolute loss of Mn2+ inhibiti on, Inhibition by high Mn2+ concentrations is shown to be due to a reductio n in k(cat); this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" m odel explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory .