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 (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 KC1 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. 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. © 1989, American Chemical Society. All rights reserved.