Hydrogen-Tritium Exchange Titration of the Histidine Residues in Ribonuclease T1 and Analysis of Their Microenvironments1

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Abstract
The overall reaction rates of hydrogen-tritium exchange occurring at the C–1positions of the imidazole rings of the three histidine residues located at positions 27, 40, and 92 from the amino terminus of ribonuclease T1 were measured as a function of pH at 37°C and 0.2 ionic strength. From the midpoints of the sigmoid curves showing the correlation of the rate constants for the three histidine residues with pH, the following pKa values could be estimated: His-27, 7.3; His-40, 7.7; and His-92, 7.5. These high pka values suggest that the three histidine residues interact strongly with neighboring negatively charged groups in the three-dimensional structure of the enzyme in solution. The sigmoid curves for His-40 and His-92 obtained in tritium exchange titration in the presence of a competitive inhibitor, 3'-GMP, shifted to alkaline pH compared with those obtained in the absence of the inhibitor, suggesting that the two histidine residues participate in the active site of the enzyme. The magnitudes of the second-order rate constants for the three histidines in the exchange reaction in the absence of the inhibitor suggest that His-27 and His-92 are embedded in the molecule to a similar extent and that His-40 is exposed on the molecular surface of the enzyme. It is possible to deduce features of the microenvironments around individual histidine residues in the enzyme molecule based on the results mentioned above. Histidine-40, which interacts strongly with a carboxylate group, is located near the entrance of the active site crevice and is exposed to the outer environment, His-92 is located at or near the bottom of the active site crevice and interacts with the carboxylate group of an acidic amino acid residue, while His-27 is not involved directly in the formation of the active site and is located in a hydrophobic region, interacting with a negatively charged carboxyl group. The tritium exchange profiles for the three histidines based on the results obtained in the absence and presence of 3'-GMP in the present study provide information necessary for assignment of the C–1proton NMR peaks due to the three histidine residues in ribonuclease T1; measurements of these were carried out previously.

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