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 that catalyze the hydrolysis of the 3'-O---P bond of RNA in RNA.DNA hybrids. The mechanism by which RNases H use active-site metal(s) for catalysis is unclear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HIV RNase H domain, two models explaining RNase H metal dependence have been proposed: a one-metal mechanism and a two-metal mechanism. In this paper, we show that the Mn2+-dependent activity of E. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the presence 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. Neutralization of His-124 by mutation to Ala results in an enzyme with a significantly decreased specific activity and an absolute loss of Mn2+ inhibition. Inhibition by high Mn2+ concentrations is shown to be due to a reduction in kcat; this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" model explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory.
Keck
J L
JL
Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Goedken
E R
ER
Marqusee
S
S
D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D015497:HIV-1 / Q000201:enzymology*; D006639:Histidine; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D008345:Manganese; D006639:Histidine; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI