Hydrogen-1 nuclear magnetic resonance studies of staphylococcal nuclease variant H124L: pH dependence of histidines and tyrosines.
The pH dependence of the 1H NMR spectrum of staphylococcal nuclease H124L was investigated as a function of the binding of Ca2+, the ion required for enzymatic activity, and deoxythymidine-3',5'-diphosphate (pdTp), a competitive inhibitor. The protein studied was the product of a cloned gene expressed in Escherichia coli which yields a protein having a sequence identical to that of the nuclease isolated from the V8 strain of Staphylococcus aureus. Of the observable ring protons of the three histidine residues, only the C delta 1H of His46 shows a large chemical shift perturbation on formation of the ternary complex, (nuclease H124L).pdTp.Ca2+. The pKa of His46 is lowered by 0.2 pH unit in the binary complex. All seven tyrosines titrate with normal pKa values between 9 and 11 in the unligated nuclease. In the ternary complex, however, the pKa values of Tyr85 and Tyr93 increase above pH 11.0. The chemical shift perturbations of the ring protons of the Tyr27, Tyr85, Tyr113, and Tyr115 were observed between pH 4 and 6; these spectral perturbations are attributed to interactions with carboxylate groups. Binding Ca2+ alone acted opposite to the perturbation in Tyr113 and Tyr115. Ca2+ binding leads to deshielding the ring protons of Tyr113, but this effect is removed in the ternary complex. Binding of pdTp and Ca2+ stabilizes the protein against high pH denaturation up to pH 11.5.
Chinami
M
M
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison 53706.
Shingu
M
M
D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013223:Statistics as Topic; D013942:Thymine Nucleotides / Q000494:pharmacology; D014443:Tyrosine / Q000032:analysis*
D013942:Thymine Nucleotides; D014443:Tyrosine; C027923:thymidine 5'-diphosphate; D006639:Histidine; D008836:Micrococcal Nuclease; D002118:Calcium