Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Jul;77(1):85–98. doi: 10.1016/S0006-3495(99)76874-8

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

E Y Lau 1, T C Bruice 1
PMCID: PMC1300314  PMID: 10388742

Abstract

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.

Full Text

The Full Text of this article is available as a PDF (379.2 KB).

Selected References

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

  1. Ash E. L., Sudmeier J. L., De Fabo E. C., Bachovchin W. W. A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment. Science. 1997 Nov 7;278(5340):1128–1132. doi: 10.1126/science.278.5340.1128. [DOI] [PubMed] [Google Scholar]
  2. Bachovchin W. W., Kaiser R., Richards J. H., Roberts J. D. Catalytic mechanism of serine proteases: reexamination of the pH dependence of the histidyl 1J13C2-H coupling constant in the catalytic triad of alpha-lytic protease. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7323–7326. doi: 10.1073/pnas.78.12.7323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blow D. M., Birktoft J. J., Hartley B. S. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature. 1969 Jan 25;221(5178):337–340. doi: 10.1038/221337a0. [DOI] [PubMed] [Google Scholar]
  4. Bruice T. C. Some pertinent aspects of mechanism as determined with small molecules. Annu Rev Biochem. 1976;45:331–373. doi: 10.1146/annurev.bi.45.070176.001555. [DOI] [PubMed] [Google Scholar]
  5. Chandrasekhar I., Clore G. M., Szabo A., Gronenborn A. M., Brooks B. R. A 500 ps molecular dynamics simulation study of interleukin-1 beta in water. Correlation with nuclear magnetic resonance spectroscopy and crystallography. J Mol Biol. 1992 Jul 5;226(1):239–250. doi: 10.1016/0022-2836(92)90136-8. [DOI] [PubMed] [Google Scholar]
  6. Cleland W. W., Kreevoy M. M. Low-barrier hydrogen bonds and enzymic catalysis. Science. 1994 Jun 24;264(5167):1887–1890. doi: 10.1126/science.8009219. [DOI] [PubMed] [Google Scholar]
  7. Creveld L. D., Amadei A., van Schaik R. C., Pepermans H. A., de Vlieg J., Berendsen H. J. Identification of functional and unfolding motions of cutinase as obtained from molecular dynamics computer simulations. Proteins. 1998 Nov 1;33(2):253–264. [PubMed] [Google Scholar]
  8. Cygler M., Schrag J. D. Structure as basis for understanding interfacial properties of lipases. Methods Enzymol. 1997;284:3–27. doi: 10.1016/s0076-6879(97)84003-7. [DOI] [PubMed] [Google Scholar]
  9. Fersht A. R., Sperling J. The charge relay system in chymotrypsin and chymotrypsinogen. J Mol Biol. 1973 Feb 25;74(2):137–149. doi: 10.1016/0022-2836(73)90103-4. [DOI] [PubMed] [Google Scholar]
  10. Frey P. A., Whitt S. A., Tobin J. B. A low-barrier hydrogen bond in the catalytic triad of serine proteases. Science. 1994 Jun 24;264(5167):1927–1930. doi: 10.1126/science.7661899. [DOI] [PubMed] [Google Scholar]
  11. Gerlt J. A., Kreevoy M. M., Cleland W., Frey P. A. Understanding enzymic catalysis: the importance of short, strong hydrogen bonds. Chem Biol. 1997 Apr;4(4):259–267. doi: 10.1016/s1074-5521(97)90069-7. [DOI] [PubMed] [Google Scholar]
  12. Guthrie J. P. Short strong hydrogen bonds: can they explain enzymic catalysis? Chem Biol. 1996 Mar;3(3):163–170. doi: 10.1016/s1074-5521(96)90258-6. [DOI] [PubMed] [Google Scholar]
  13. Hedstrom L., Szilagyi L., Rutter W. J. Converting trypsin to chymotrypsin: the role of surface loops. Science. 1992 Mar 6;255(5049):1249–1253. doi: 10.1126/science.1546324. [DOI] [PubMed] [Google Scholar]
  14. Kraut J. Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem. 1977;46:331–358. doi: 10.1146/annurev.bi.46.070177.001555. [DOI] [PubMed] [Google Scholar]
  15. Longhi S., Czjzek M., Lamzin V., Nicolas A., Cambillau C. Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis. J Mol Biol. 1997 May 16;268(4):779–799. doi: 10.1006/jmbi.1997.1000. [DOI] [PubMed] [Google Scholar]
  16. Longhi S., Nicolas A., Creveld L., Egmond M., Verrips C. T., de Vlieg J., Martinez C., Cambillau C. Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants. Proteins. 1996 Dec;26(4):442–458. doi: 10.1002/(SICI)1097-0134(199612)26:4<442::AID-PROT5>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  17. Martinez C., Nicolas A., van Tilbeurgh H., Egloff M. P., Cudrey C., Verger R., Cambillau C. Cutinase, a lipolytic enzyme with a preformed oxyanion hole. Biochemistry. 1994 Jan 11;33(1):83–89. doi: 10.1021/bi00167a011. [DOI] [PubMed] [Google Scholar]
  18. Nicolas A., Egmond M., Verrips C. T., de Vlieg J., Longhi S., Cambillau C., Martinez C. Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state. Biochemistry. 1996 Jan 16;35(2):398–410. doi: 10.1021/bi9515578. [DOI] [PubMed] [Google Scholar]
  19. Ohmae E., Iriyama K., Ichihara S., Gekko K. Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function. J Biochem. 1996 Apr;119(4):703–710. doi: 10.1093/oxfordjournals.jbchem.a021299. [DOI] [PubMed] [Google Scholar]
  20. Philippopoulos M., Lim C. Molecular dynamics simulation of E. coli ribonuclease H1 in solution: correlation with NMR and X-ray data and insights into biological function. J Mol Biol. 1995 Dec 8;254(4):771–792. doi: 10.1006/jmbi.1995.0654. [DOI] [PubMed] [Google Scholar]
  21. Prompers J. J., Groenewegen A., Van Schaik R. C., Pepermans H. A., Hilbers C. W. 1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution. Protein Sci. 1997 Nov;6(11):2375–2384. doi: 10.1002/pro.5560061111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Quirk D. J., Park C., Thompson J. E., Raines R. T. His...Asp catalytic dyad of ribonuclease A: conformational stability of the wild-type, D121N, D121A, and H119A enzymes. Biochemistry. 1998 Dec 22;37(51):17958–17964. doi: 10.1021/bi981688j. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Schrag J. D., Cygler M. Lipases and alpha/beta hydrolase fold. Methods Enzymol. 1997;284:85–107. doi: 10.1016/s0076-6879(97)84006-2. [DOI] [PubMed] [Google Scholar]
  25. Steitz T. A., Shulman R. G. Crystallographic and NMR studies of the serine proteases. Annu Rev Biophys Bioeng. 1982;11:419–444. doi: 10.1146/annurev.bb.11.060182.002223. [DOI] [PubMed] [Google Scholar]
  26. Verma C. S., Caves L. S., Hubbard R. E., Roberts G. C. Domain motions in dihydrofolate reductase: a molecular dynamics study. J Mol Biol. 1997 Mar 7;266(4):776–796. doi: 10.1006/jmbi.1996.0818. [DOI] [PubMed] [Google Scholar]
  27. Warshel A. Electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites. J Biol Chem. 1998 Oct 16;273(42):27035–27038. doi: 10.1074/jbc.273.42.27035. [DOI] [PubMed] [Google Scholar]
  28. Warshel A., Florián J. Computer simulations of enzyme catalysis: finding out what has been optimized by evolution. Proc Natl Acad Sci U S A. 1998 May 26;95(11):5950–5955. doi: 10.1073/pnas.95.11.5950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Warshel A., Papazyan A. Energy considerations show that low-barrier hydrogen bonds do not offer a catalytic advantage over ordinary hydrogen bonds. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13665–13670. doi: 10.1073/pnas.93.24.13665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Warshel A., Papazyan A., Kollman P. A. On low-barrier hydrogen bonds and enzyme catalysis. Science. 1995 Jul 7;269(5220):102–106. doi: 10.1126/science.7661987. [DOI] [PubMed] [Google Scholar]
  32. Warshel A., Sussman F., Hwang J. K. Evaluation of catalytic free energies in genetically modified proteins. J Mol Biol. 1988 May 5;201(1):139–159. doi: 10.1016/0022-2836(88)90445-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES