Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry.
Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates. Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed. Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT. Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results. However, only one Mg2+ ion is bound in the RNase H catalytic center. Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain. The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported.
Cowan
J A
JA
Evans Laboratory of Chemistry, The Ohio State University, Columbus 43210, USA. cowan@chemistry.ohio-state.edu
Ohyama
T
T
Howard
K
K
Rausch
J W
JW
Cowan
S M
SM
Le Grice
S F
SF
D002151:Calorimetry; D018360:Crystallography, X-Ray; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D009696:Nucleic Acids / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013816:Thermodynamics
D009696:Nucleic Acids; D011994:Recombinant Proteins; D008345:Manganese; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H