Hydrogen-Tritium Exchange and Nuclear Magnetic Resonance Titrations of the Histidine Residues in Ribonuclease St and Analysis of Their Microenvironment1

Abstract

Ribonuclease St consists of 101 amino acid residues in a single polypeptide chain with one disulfide bond. It has two histidines lccated at positions 60 and 91 from the amino terminus. The pK₂, values of His-60 and His-91 were estimated by hydrogen-tritium exchange titration to be 8.0 and 6.3, respectively, and these values were confirmed by ¹H NMR titration. The high pK₂ value of His-60 suggests that it interacts with a neighboring negative charge, presumably of a carboxylate. This is suggested by the presence of an inflection at pH 4.5 in the ¹H NMR titration plot for His-60. The ¹H NMR titration plot for His-91 also suggests its interaction with a carboxylate, although the pK₂ of His-91 was close to that of unperturbed histidine residues. This suggests that, a positively charged group is also located in the vicinity of His-91. It was concluded that His-91 is one of the active site residues of the enzyme. The pK₂, for His-91 was shifted to the alkaline side in the presence of 3'-GMP, a competitive inhibitor, in the titration plots observed by both hydrogen-tritium exchange and ¹H NMR spectroscopy. The ³¹P NMR titration data suggest that in the 3'-GMP-RNase St complex the dianion form of the nucleotide participates in the interaction with the protonated form of His-91. The existence of another positively charged group with pK₂, of 7.0 was also suggested on the basis of the ³¹P NMR data. The hydrogen-tritium exchange rate constants for the two histidines suggest that His-60 is embeded slightly in the molecule, while His-91 is exposed to the solvent. The present results are discussed briefly in terms of preliminary results of X-ray structural studies which are in progress.