Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Sep 15;88(18):8116–8120. doi: 10.1073/pnas.88.18.8116

Histidine pKa shifts accompanying the inactivating Asp121----Asn substitution in a semisynthetic bovine pancreatic ribonuclease.

M T Cederholm 1, J A Stuckey 1, M S Doscher 1, L Lee 1
PMCID: PMC52457  PMID: 1896458

Abstract

A semisynthetic RNase, RNase-(1-118).(111-124), consisting of a noncovalent complex between residues 1-118 of RNase (obtained from the proteolytic digestion of RNase A), and a synthetic 14-residue peptide containing residues 111-124 of RNase, exhibits 98% of the enzymatic activity of bovine pancreatic ribonuclease A (EC 3.1.27.5). The replacement of aspartic acid-121 by asparagine in this semisynthetic RNase to form the "D121N" analog reduces kcat/Km to 2.7% of the value for RNase A. In the present work, 1H NMR spectroscopy has been used to probe the ionization states of His12, His105, and His119 in this catalytically defective semisynthetic RNase. A comparison of the observed resonances of D121N with those previously determined by others for RNase A enabled us to assign the C2 proton NMR resonances to individual residues; the assignment of His119 was confirmed by titrating D121N with the fully deuterated peptide, [Asn121]-RNase-(111-124). The observed pKa values of His12, His105, and His119 decrease 0.18, 0.16, and 0.02 pH unit, respectively, as a result of the D121N replacement. Values calculated by using a finite difference algorithm to solve the Poisson-Boltzmann equation (the DELPHI program, version 3.0) and a refined 2.0-A coordinate set for the crystal structure of D121N differ significantly for active site residues His12 (delta pKa = -0.58) and His119 (delta pKa = -0.55) but not for His105 (delta pKa = -0.10). The elmination of bound water from the calculations reduced, but did not reconcile, these discrepancies (His12, delta pKa = -0.36; His119, delta pKa = -0.41).

Full text

PDF
8116

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beintema J. J., Schüller C., Irie M., Carsana A. Molecular evolution of the ribonuclease superfamily. Prog Biophys Mol Biol. 1988;51(3):165–192. doi: 10.1016/0079-6107(88)90001-6. [DOI] [PubMed] [Google Scholar]
  2. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  3. Bradbury J. H., Crompton M. W., Teh J. S. Nuclear-magnetic-resonance study of the histidine residues of S-peptide and S-protein and kinetics of 1H-2H exchange of ribonuclease A. Eur J Biochem. 1977 Dec 1;81(2):411–422. doi: 10.1111/j.1432-1033.1977.tb11966.x. [DOI] [PubMed] [Google Scholar]
  4. Craik C. S., Roczniak S., Largman C., Rutter W. J. The catalytic role of the active site aspartic acid in serine proteases. Science. 1987 Aug 21;237(4817):909–913. doi: 10.1126/science.3303334. [DOI] [PubMed] [Google Scholar]
  5. Doscher M. S., Martin P. D., Edwards B. F. Characterization of the histidine proton nuclear magnetic resonances of a semisynthetic ribonuclease. Biochemistry. 1983 Aug 16;22(17):4125–4131. doi: 10.1021/bi00286a021. [DOI] [PubMed] [Google Scholar]
  6. Gilson M. K., Honig B. H. Calculation of electrostatic potentials in an enzyme active site. Nature. 1987 Nov 5;330(6143):84–86. doi: 10.1038/330084a0. [DOI] [PubMed] [Google Scholar]
  7. Gilson M. K., Honig B. H. Energetics of charge-charge interactions in proteins. Proteins. 1988;3(1):32–52. doi: 10.1002/prot.340030104. [DOI] [PubMed] [Google Scholar]
  8. Gutte B., Lin M. C., Caldi D. G., Merrifield R. B. Reactivation of des(119-, 120-, or 121-124) ribonuclease A by mixture with synthetic COOH-terminal peptides of varying lengths. J Biol Chem. 1972 Aug 10;247(15):4763–4767. [PubMed] [Google Scholar]
  9. Klapper I., Hagstrom R., Fine R., Sharp K., Honig B. Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins. 1986 Sep;1(1):47–59. doi: 10.1002/prot.340010109. [DOI] [PubMed] [Google Scholar]
  10. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  11. Lin M. C., Gutte B., Moore S., Merrifield R. B. Regeneration of activity by mixture of ribonuclease enzymically degraded from the COOH terminus and a synthetic COOH-terminal tetradecapeptide. J Biol Chem. 1970 Oct 10;245(19):5169–5170. [PubMed] [Google Scholar]
  12. Lin M. C. The structural roles of amino acid residues near the carboxyl terminus of bovine pancreatic ribonuclease A. J Biol Chem. 1970 Dec 25;245(24):6726–6731. [PubMed] [Google Scholar]
  13. Markley J. L. Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments. Biochemistry. 1975 Aug 12;14(16):3546–3554. doi: 10.1021/bi00687a006. [DOI] [PubMed] [Google Scholar]
  14. Markley J. L. Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments. Biochemistry. 1975 Aug 12;14(16):3546–3554. doi: 10.1021/bi00687a006. [DOI] [PubMed] [Google Scholar]
  15. Markley J. L. Nuclear magnetic resonance studies of trypsin inhibitors. Histidines of virgin and modified soybean trypsin inhibitor (Kunitz). Biochemistry. 1973 Jun 5;12(12):2245–2250. doi: 10.1021/bi00736a010. [DOI] [PubMed] [Google Scholar]
  16. Martin P. D., Doscher M. S., Edwards B. F. The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution. J Biol Chem. 1987 Nov 25;262(33):15930–15938. doi: 10.2210/pdb1srn/pdb. [DOI] [PubMed] [Google Scholar]
  17. Meadows D. H., Jardetzky O., Epand R. M., Ruterjans H. H., Scheraga H. A. Assignment of the histidine peaks in the nuclear magnetic resonance spectrum of ribonuclease. Proc Natl Acad Sci U S A. 1968 Jul;60(3):766–772. doi: 10.1073/pnas.60.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Merrifield B. Solid phase synthesis. Science. 1986 Apr 18;232(4748):341–347. doi: 10.1126/science.3961484. [DOI] [PubMed] [Google Scholar]
  19. Patel D. J., Canuel L. L., Bovey F. A. Reassignment of the active site histidines in ribonuclease A by selective deuteration studies. Biopolymers. 1975 May;14(5):987–997. doi: 10.1002/bip.1975.360140508. [DOI] [PubMed] [Google Scholar]
  20. Richards F. M. Calculation of molecular volumes and areas for structures of known geometry. Methods Enzymol. 1985;115:440–464. doi: 10.1016/0076-6879(85)15032-9. [DOI] [PubMed] [Google Scholar]
  21. Roberts G. C., Meadows D. H., Jardetzky O. Nuclear magnetic resonance studies of the sx.ucture and binding sites of enzymes. VII. Solvent and temperature effects on the ionization of histidine residues of ribonuclease. Biochemistry. 1969 May;8(5):2053–2056. doi: 10.1021/bi00833a040. [DOI] [PubMed] [Google Scholar]
  22. Russell A. J., Thomas P. G., Fersht A. R. Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering. J Mol Biol. 1987 Feb 20;193(4):803–813. doi: 10.1016/0022-2836(87)90360-3. [DOI] [PubMed] [Google Scholar]
  23. Sharp K. A., Honig B. Electrostatic interactions in macromolecules: theory and applications. Annu Rev Biophys Biophys Chem. 1990;19:301–332. doi: 10.1146/annurev.bb.19.060190.001505. [DOI] [PubMed] [Google Scholar]
  24. Shindo H., Hayes M. B., Cohen J. S. Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease. J Biol Chem. 1976 May 10;251(9):2644–2647. [PubMed] [Google Scholar]
  25. Sprang S., Standing T., Fletterick R. J., Stroud R. M., Finer-Moore J., Xuong N. H., Hamlin R., Rutter W. J., Craik C. S. The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. Science. 1987 Aug 21;237(4817):905–909. doi: 10.1126/science.3112942. [DOI] [PubMed] [Google Scholar]
  26. Stern M. S., Doscher M. S. Aspartic acid-121 functions at the active site of bovine pancreatic ribonuclease. FEBS Lett. 1984 Jun 11;171(2):253–256. doi: 10.1016/0014-5793(84)80498-6. [DOI] [PubMed] [Google Scholar]
  27. Sternberg M. J., Hayes F. R., Russell A. J., Thomas P. G., Fersht A. R. Prediction of electrostatic effects of engineering of protein charges. Nature. 1987 Nov 5;330(6143):86–88. doi: 10.1038/330086a0. [DOI] [PubMed] [Google Scholar]
  28. Sun D. P., Liao D. I., Remington S. J. Electrostatic fields in the active sites of lysozymes. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5361–5365. doi: 10.1073/pnas.86.14.5361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wlodawer A., Bott R., Sjölin L. The refined crystal structure of ribonuclease A at 2.0 A resolution. J Biol Chem. 1982 Feb 10;257(3):1325–1332. [PubMed] [Google Scholar]
  30. Wlodawer A., Sjölin L. Orientation of histidine residues in RNase A: neutron diffraction study. Proc Natl Acad Sci U S A. 1981 May;78(5):2853–2855. doi: 10.1073/pnas.78.5.2853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wlodawer A., Sjölin L. Structure of ribonuclease A: results of joint neutron and X-ray refinement at 2.0-A resolution. Biochemistry. 1983 May 24;22(11):2720–2728. doi: 10.1021/bi00280a021. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES