Staphylococcal nuclease active-site amino acids: pH dependence of tyrosines and arginines by carbon-13 NMR and correlation with kinetic studies

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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 (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-. 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 infection 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. 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 protein was enriched uniformly with 20% 13C at all carbons and specifically with 90% 13C at the guanidino carbon of the arginine residues. All five arginines in the protein have pKa values greater than 11.6 in H2O and 0.3 M KCl. This eliminates arginine as a candidate for the basic catalyst that deprotonates H2O to facilitate nucleophilic attack on phosphorus. 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. The structure of nuclease Y85F is not perturbed significantly by this substitution. Tyrosine-85 has a spectroscopically observed pKa of 9.53 .+-. 0.05 in H2O and 0.3 M KCl, which is similar to the basic-side pKa of 9.67 seen in the kcat/Km pH profile. 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''-phosphomonoester. 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.