Characterization of pKa Values and Titration Shifts in the Cytotoxic Ribonuclease -Sarcin by NMR. Relationship bet
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- 作者：José ; Manuel Pé ; rez-Cañ ; adillas ; Ramó ; n Campos-Olivas ; Javier Lacadena ; Alvaro Martí ; nez del Pozo ; José ; G. Gavilanes ; Jorge Santoro ; Manuel Rico ; and Marta Bruix
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：November 10, 1998
- 年：1998
- 卷：37
- 期：45
- 页码：15865 - 15876
- 全文大小：171K
- 年卷期：v.37,no.45(November 10, 1998)
- ISSN：1520-4995
文摘
The electrostatic behavior of titrating groups in 
-sarcin was investigated using 1H NMRspectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in themolecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data tosimple relationships derived from the Henderson-Hasselbalch equation led to the unambiguousdetermination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminalcarboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalyticallyrelevant histidines, His50 and His137, and glutamic acid, Glu96, in the -sarcin-2'-GMP complex werealso determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamicacids, and four histidines) were found to be close to their normal values. On the other hand, a numberof side chain groups, including those in the active center, showed pKa values far from their intrinsicvalues. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 inthe free enzyme and 7.6, ~4.8, and 6.8 in the -sarcin-2'-GMP complex, respectively. The pKa valuesand the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymaticmechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, aPhe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137).The absence of this interaction in -sarcin would explain the lower pKa value of this His residue, andprovides an explanation for the decreased RNase activity of this protein as compared to those of othermicrobial ribonucleases.
-sarcin was investigated using 1H NMRspectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in themolecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data tosimple relationships derived from the Henderson-Hasselbalch equation led to the unambiguousdetermination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminalcarboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalyticallyrelevant histidines, His50 and His137, and glutamic acid, Glu96, in the -sarcin-2'-GMP complex werealso determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamicacids, and four histidines) were found to be close to their normal values. On the other hand, a numberof side chain groups, including those in the active center, showed pKa values far from their intrinsicvalues. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 inthe free enzyme and 7.6, ~4.8, and 6.8 in the -sarcin-2'-GMP complex, respectively. The pKa valuesand the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymaticmechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, aPhe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137).The absence of this interaction in -sarcin would explain the lower pKa value of this His residue, andprovides an explanation for the decreased RNase activity of this protein as compared to those of othermicrobial ribonucleases.