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. 1992 Sep 15;286(Pt 3):889–900. doi: 10.1042/bj2860889

A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin.

M D Finucane 1, J P Malthouse 1
PMCID: PMC1132987  PMID: 1417749

Abstract

delta-Chymotrypsin has been alkylated by 1-13C- and 2-13C-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-13C- and 2-13C-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pKa values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-13C- and 2-13C-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-13C-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pKa of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the 13C titration shifts of these model compounds with those of the 13C enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that, in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10,000-fold greater than it would be in water. We conclude that the pKa of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pKa of the imidazolium cation of histidine-57 is greater than 7.9 and greater than 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pKa of the imidazolium cation is greater than 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pKa of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Asbóth B., Polgár L. Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: hydrolyses of thiono and oxygen esters. Biochemistry. 1983 Jan 4;22(1):117–122. doi: 10.1021/bi00270a017. [DOI] [PubMed] [Google Scholar]
  2. Bachovchin W. W. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry. 1986 Nov 18;25(23):7751–7759. doi: 10.1021/bi00371a070. [DOI] [PubMed] [Google Scholar]
  3. Bachovchin W. W., Wong W. Y., Farr-Jones S., Shenvi A. B., Kettner C. A. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes. Biochemistry. 1988 Oct 4;27(20):7689–7697. doi: 10.1021/bi00420a018. [DOI] [PubMed] [Google Scholar]
  4. Brady K., Liang T. C., Abeles R. H. pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone. Biochemistry. 1989 Nov 14;28(23):9066–9070. doi: 10.1021/bi00449a017. [DOI] [PubMed] [Google Scholar]
  5. Brady K., Wei A. Z., Ringe D., Abeles R. H. Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors. Biochemistry. 1990 Aug 21;29(33):7600–7607. doi: 10.1021/bi00485a009. [DOI] [PubMed] [Google Scholar]
  6. Bryan P., Pantoliano M. W., Quill S. G., Hsiao H. Y., Poulos T. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3743–3745. doi: 10.1073/pnas.83.11.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter P., Wells J. A. Functional interaction among catalytic residues in subtilisin BPN'. Proteins. 1990;7(4):335–342. doi: 10.1002/prot.340070405. [DOI] [PubMed] [Google Scholar]
  8. Coggins J. R., Kray W., Shaw E. Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone. Biochem J. 1974 Mar;137(3):579–585. doi: 10.1042/bj1370579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fastrez J., Fersht A. R. Mechanism of chymotrypsin. Structure, reactivity, and nonproductive binding relationships. Biochemistry. 1973 Mar 13;12(6):1067–1074. doi: 10.1021/bi00730a008. [DOI] [PubMed] [Google Scholar]
  10. Fastrez J. On the stability of tetrahedral intermediates within the active sites of serine and cysteine proteases. Eur J Biochem. 1983 Sep 15;135(2):339–341. doi: 10.1111/j.1432-1033.1983.tb07659.x. [DOI] [PubMed] [Google Scholar]
  11. Fersht A. R., Renard M. pH dependence of chymotrypsin catalysis. Appendix: substrate binding to dimeric alpha-chymotrypsin studied by x-ray diffraction and the equilibrium method. Biochemistry. 1974 Mar 26;13(7):1416–1426. doi: 10.1021/bi00704a016. [DOI] [PubMed] [Google Scholar]
  12. Fersht A. R., Shi J. P., Knill-Jones J., Lowe D. M., Wilkinson A. J., Blow D. M., Brick P., Carter P., Waye M. M., Winter G. Hydrogen bonding and biological specificity analysed by protein engineering. Nature. 1985 Mar 21;314(6008):235–238. doi: 10.1038/314235a0. [DOI] [PubMed] [Google Scholar]
  13. Finucane M. D., Hudson E. A., Malthouse J. P. A 13C-n.m.r. investigation of the ionizations within an inhibitor--alpha-chymotrypsin complex. Evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms. Biochem J. 1989 Mar 15;258(3):853–859. doi: 10.1042/bj2580853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henderson R. Structure of crystalline alpha-chymotrypsin. IV. The structure of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme. J Mol Biol. 1970 Dec 14;54(2):341–354. doi: 10.1016/0022-2836(70)90434-1. [DOI] [PubMed] [Google Scholar]
  15. James M. N., Sielecki A. R. Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin. Biochemistry. 1985 Jul 2;24(14):3701–3713. doi: 10.1021/bi00335a045. [DOI] [PubMed] [Google Scholar]
  16. Komiyama M., Bender M. L. Do cleavages of amides by serine proteases occur through a stepwise pathway involving tetrahedral intermediates? Proc Natl Acad Sci U S A. 1979 Feb;76(2):557–560. doi: 10.1073/pnas.76.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Komiyama M., Bender M. L., Utaka M., Takeda A. Model for "charge-relay": acceleration by carboxylate anion in intramolecular general base-catalyzed ester hydrolysis by the imidazolyl group. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2634–2638. doi: 10.1073/pnas.74.7.2634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kossiakoff A. A., Spencer S. A. Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases: neutron structure of trypsin. Biochemistry. 1981 Oct 27;20(22):6462–6474. doi: 10.1021/bi00525a027. [DOI] [PubMed] [Google Scholar]
  19. Liang T. C., Abeles R. H. Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy. Biochemistry. 1987 Dec 1;26(24):7603–7608. doi: 10.1021/bi00398a011. [DOI] [PubMed] [Google Scholar]
  20. Malthouse J. P., Finucane M. D. A study of the relaxation parameters of a 13C-enriched methylene carbon and a 13C-enriched perdeuteromethylene carbon attached to chymotrypsin. Biochem J. 1991 Dec 15;280(Pt 3):649–657. doi: 10.1042/bj2800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Malthouse J. P., Primrose W. U., Mackenzie N. E., Scott A. I. 13C NMR study of the ionizations within a trypsin-chloromethyl ketone inhibitor complex. Biochemistry. 1985 Jul 2;24(14):3478–3487. doi: 10.1021/bi00335a014. [DOI] [PubMed] [Google Scholar]
  22. ONG E. B., SHAW E., SCHOELLMANN G. THE IDENTIFICATION OF THE HISTIDINE RESIDUE AT THE ACTIVE CENTER OF CHYMOTRYPSIN. J Biol Chem. 1965 Feb;240:694–698. [PubMed] [Google Scholar]
  23. Ortiz C., Tellier C., Williams H., Stolowich N. J., Scott A. I. Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy. Biochemistry. 1991 Oct 15;30(41):10026–10034. doi: 10.1021/bi00105a030. [DOI] [PubMed] [Google Scholar]
  24. Primrose W. U., Scott A. I., Mackenzie N. E., Malthouse J. P. A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion. Biochem J. 1985 Nov 1;231(3):677–682. doi: 10.1042/bj2310677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rich D. H. Pepstatin-derived inhibitors of aspartic proteinases. A close look at an apparent transition-state analogue inhibitor. J Med Chem. 1985 Mar;28(3):263–273. doi: 10.1021/jm00381a001. [DOI] [PubMed] [Google Scholar]
  26. Robillard G., Shulman R. G. High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system. J Mol Biol. 1974 Jul 5;86(3):519–540. doi: 10.1016/0022-2836(74)90178-8. [DOI] [PubMed] [Google Scholar]
  27. Robillard G., Shulman R. G. High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors. J Mol Biol. 1974 Jul 5;86(3):541–558. doi: 10.1016/0022-2836(74)90179-x. [DOI] [PubMed] [Google Scholar]
  28. Robillard G., Shulman R. G. High resolution nuclear magnetic resonance study of the histidine--aspartate hydrogen bond in chymotrypsin and chymotrypsinogen. J Mol Biol. 1972 Nov 14;71(2):507–511. doi: 10.1016/0022-2836(72)90366-x. [DOI] [PubMed] [Google Scholar]
  29. Rogers G. A., Bruice T. C. Synthesis and evaluation of a model for the so-called "charge-relay" system of the serine esterases. J Am Chem Soc. 1974 Apr 17;96(8):2473–2481. doi: 10.1021/ja00815a028. [DOI] [PubMed] [Google Scholar]
  30. Russell S. T., Warshel A. Calculations of electrostatic energies in proteins. The energetics of ionized groups in bovine pancreatic trypsin inhibitor. J Mol Biol. 1985 Sep 20;185(2):389–404. doi: 10.1016/0022-2836(85)90411-5. [DOI] [PubMed] [Google Scholar]
  31. SCHOELLMANN G., SHAW E. Direct evidence for the presence of histidine in the active center of chymotrypsin. Biochemistry. 1963 Mar-Apr;2:252–255. doi: 10.1021/bi00902a008. [DOI] [PubMed] [Google Scholar]
  32. Satterthwait A. C., Jencks W. P. The mechanism of the aminolysis of acetate esters. J Am Chem Soc. 1974 Oct 30;96(22):7018–7031. doi: 10.1021/ja00829a034. [DOI] [PubMed] [Google Scholar]
  33. Shaw E., Springhorn S. Identification of the histidine residue at the active center of trypsin labelled by TLCK. Biochem Biophys Res Commun. 1967 May 5;27(3):391–397. doi: 10.1016/s0006-291x(67)80112-8. [DOI] [PubMed] [Google Scholar]
  34. Spomer W. E., Wootton J. F. The hydrolysis of alpha-N-benzoyl-L-argininamide catalyzed by trypsin and acetyltrypsin. Dependence on pH. Biochim Biophys Acta. 1971 Apr 14;235(1):164–171. doi: 10.1016/0005-2744(71)90044-1. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Warshel A. Energetics of enzyme catalysis. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5250–5254. doi: 10.1073/pnas.75.11.5250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Warshel A., Naray-Szabo G., Sussman F., Hwang J. K. How do serine proteases really work? Biochemistry. 1989 May 2;28(9):3629–3637. doi: 10.1021/bi00435a001. [DOI] [PubMed] [Google Scholar]
  38. Warshel A., Russell S. T. Calculations of electrostatic interactions in biological systems and in solutions. Q Rev Biophys. 1984 Aug;17(3):283–422. doi: 10.1017/s0033583500005333. [DOI] [PubMed] [Google Scholar]
  39. Warshel A., Russell S. T., Churg A. K. Macroscopic models for studies of electrostatic interactions in proteins: limitations and applicability. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4785–4789. doi: 10.1073/pnas.81.15.4785. [DOI] [PMC free article] [PubMed] [Google Scholar]

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