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 have been examined with three p-nitrophenyl phosphate containing DNA analogues that vary as to 3'-substituent. With wild-type (Foggi variant) nuclease (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. The data suggests that the inflection kcat/Km with pKa at 9.67 arises from ionization of tyrosine-85, which hydrogen bonds to the divalent 3'-phosphomonester of substrates with this substituent. The enthalpy of ionization of both deprotonation steps in the kcat/Km versus pH profile is 5 kcal/mol. 13C NMR has been used to determine the pKa values of the arginine and tyrosine residues. The results do not rule out arginine as a candidate for the acidic catalyst that protonates the 5'-ribose alkoxide prior to product release. The phenolic hydroxyl carbon of tyrosine-85 has been assigned by comparing the 13C NMR spectrum of nuclease wt and nuclease Y85F. This correlation between pKa values along with the absence of other candidates indicates that the ionization of tyrosine-85 is the pKa seen in the kcat/Km vs pH profile for substrates with a divalent 3'-phosphomonester. This conclusion is consistent with the proposed role of tyrosine-85 as a hydrogen-bond donor to the 3'-phosphomonoester of substrates poised for exonucleolytic hydrolysis