title: Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies.
abstract: The pH and temperature dependence of the kinetic parameters of staphylococcal nuclease (EC 3.1.4.7) have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) (nuclease wt) and the substrates thymidine 3'-phosphate 5'-(p-nitrophenyl phosphate) (PNPdTp), thymidine 3'-methylphosphonate 5'-(p-nitrophenyl phosphate) (PNPdTp*Me), and thymidine 5'-(p-nitrophenyl phosphate) (PNPdT), kcat remains nearly constant at 13 min-1. However, kcat/Km with nuclease wt varies considerably: 413, 13, and 0.52 mM-1 min-1 with PNPdTp, PNPdTp*Me, and PNPdT, respectively. When tyrosine-85 is changed to phenylalanine (nuclease Y85F) by site-directed mutagenesis, kcat is unchanged at about 13 min-1, except with PNPdTp where it drops to 1 min-1. With nuclease Y85F, kcat/Km is 19.5 and 25 mM-1 min-1 with PNPdTp and PNPdTp*Me, respectively. With PNPdTp as the substrate, a bell-shaped kcat/Km vs pH profile is seen with pKa values at 8.94 and 9.67 in 0.3 M KCl and H2O. The pKa at 9.67 disappears, and a new pKa appears at 10.1 when tyrosine-85 is changed to phenylalanine (nuclease Y85F) or when the substrate 3'-phosphomonoester is changed to a 3'-methylphosphonate (PNPdTp*Me). This suggests that the inflection in kcat/Km with pKa at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phosphomonoester of substrates with this substituent.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
authors.forename: C B
authors.initials: CB
authors.lastname: Grissom
authors.forename: J L
authors.initials: JL
authors.lastname: Markley
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine; D013696:Temperature; D013816:Thermodynamics; D014169:Transformation, Bacterial; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D010649:Phenylalanine; D001120:Arginine; D008836:Micrococcal Nuclease