Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by 13C NMR and correlation with kinetic studies.
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)
Grissom
C B
CB
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Markley
J L
JL
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*
D014443:Tyrosine; D010649:Phenylalanine; D001120:Arginine; D008836:Micrococcal Nuclease