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His ... Asp Catalytic Dyad of Ribonuclease A: Histidine pKa Values in the Wild-Type, D121N, and D121A Enzymes

1999; Elsevier BV; Volume: 76; Issue: 3 Linguagem: Inglês

10.1016/s0006-3495(99)77316-9

1542-0086

David J. Quirk, Ronald T. Raines,

DNA Repair Mechanisms

Abstract Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp 121 forms a hydrogen bond with His 119 , which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His 12 , which is also in the active site. Here, 1 H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp 121 on the microscopic p K a values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His 119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3′-phosphate (3′-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3′-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations 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 catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.