His - Asp catalytic dyad of ribonuclease A: Histidine pK(a) values in the wild-type, D121N, and D121A enzymes

Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp cata lytic dyad in its active site. Structural analyses had indicated that Asp(1 21) forms a hydrogen bond with His(119), which serves as an acid during cat alysis of RNA cleavage. The enzyme contains three other histidine residues including His(12), which is also in the active site. Here, H-1-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroug hly as a function of pH. The effect of replacing Asp(121) on the microscopi c pK(a) values of the histidine residues is modest: none change by more tha n 0.2 units. There is no evidence for the formation of a low-barrier hydrog en bond between His(119) and either an aspartate or an asparagine residue a t position 121. In the presence of the reaction product, uridine 3'-phospha te (3'-UMP), protonation of one active-site histidine residue favors proton ation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residu es when both are protonated, Comparison of the titration curves of the unli ganded enzyme with that obtained in the presence of different concentration s of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp c atalytic dyad of RNase A has a measurable but modest effect on the ionizati on of the adjacent histidine residue.