Hydrogen-tritium exchange and nuclear magnetic resonance titrations of the histidine residues in ribonuclease St and analysis of their microenvironment.
Ribonuclease St consists of 101 amino acid residues in a single polypeptide chain with one disulfide bond. It has two histidines located at positions 60 and 91 from the amino terminus. The pKa 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 pKa 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 pKa 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 pKa 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 pKa of 7.0 was also suggested on the basis of the 31P NMR data.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D012260:Ribonucleases; D013302:Streptomyces / Q000201:enzymology; D014316:Tritium
D000596:Amino Acids; D010446:Peptide Fragments; D014316:Tritium; D006639:Histidine; D006157:Guanosine Monophosphate; D004722:Endoribonucleases; D012260:Ribonucleases; D006163:Ribonuclease T1