Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Jul;5(7):1355–1365. doi: 10.1002/pro.5560050714

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

A J Beveridge 1
PMCID: PMC2143470  PMID: 8819168

Abstract

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Drenth J., Kalk K. H., Swen H. M. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry. 1976 Aug 24;15(17):3731–3738. doi: 10.1021/bi00662a014. [DOI] [PubMed] [Google Scholar]
  2. Garavito R. M., Rossmann M. G., Argos P., Eventoff W. Convergence of active center geometries. Biochemistry. 1977 Nov 15;16(23):5065–5071. doi: 10.1021/bi00642a019. [DOI] [PubMed] [Google Scholar]
  3. Halász P., Polgár L. Negatively charged reactants as probes in the study of the essential mercaptide-imidazolium ion-pair of thiolenzymes. Eur J Biochem. 1977 Oct 3;79(2):491–494. doi: 10.1111/j.1432-1033.1977.tb11832.x. [DOI] [PubMed] [Google Scholar]
  4. Higaki J. N., Evnin L. B., Craik C. S. Introduction of a cysteine protease active site into trypsin. Biochemistry. 1989 Nov 28;28(24):9256–9263. doi: 10.1021/bi00450a004. [DOI] [PubMed] [Google Scholar]
  5. Kamphuis I. G., Kalk K. H., Swarte M. B., Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984 Oct 25;179(2):233–256. doi: 10.1016/0022-2836(84)90467-4. [DOI] [PubMed] [Google Scholar]
  6. Lewis S. D., Johnson F. A., Shafer J. A. Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidence for a his-159--Cys-25 ion pair and its possible role in catalysis. Biochemistry. 1981 Jan 6;20(1):48–51. doi: 10.1021/bi00504a009. [DOI] [PubMed] [Google Scholar]
  7. Lewis S. D., Johnson F. A., Shafer J. A. Potentiometric determination of ionizations at the active site of papain. Biochemistry. 1976 Nov 16;15(23):5009–5017. doi: 10.1021/bi00668a010. [DOI] [PubMed] [Google Scholar]
  8. Matthews D. A., Smith W. W., Ferre R. A., Condon B., Budahazi G., Sisson W., Villafranca J. E., Janson C. A., McElroy H. E., Gribskov C. L. Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell. 1994 Jun 3;77(5):761–771. doi: 10.1016/0092-8674(94)90059-0. [DOI] [PubMed] [Google Scholar]
  9. Neet K. E., Koshland D. E., Jr The conversion of serine at the active site of subtilisin to cysteine: a "chemical mutation". Proc Natl Acad Sci U S A. 1966 Nov;56(5):1606–1611. doi: 10.1073/pnas.56.5.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pathak D., Ollis D. Refined structure of dienelactone hydrolase at 1.8 A. J Mol Biol. 1990 Jul 20;214(2):497–525. doi: 10.1016/0022-2836(90)90196-s. [DOI] [PubMed] [Google Scholar]
  11. Polgar L., Bender M. L. The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group. Biochemistry. 1967 Feb;6(2):610–620. doi: 10.1021/bi00854a032. [DOI] [PubMed] [Google Scholar]
  12. Roberts D. D., Lewis S. D., Ballou D. P., Olson S. T., Shafer J. A. Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry. 1986 Sep 23;25(19):5595–5601. doi: 10.1021/bi00367a038. [DOI] [PubMed] [Google Scholar]
  13. Rullmann J. A., Bellido M. N., van Duijnen P. T. The active site of papain. All-atom study of interactions with protein matrix and solvent. J Mol Biol. 1989 Mar 5;206(1):101–118. doi: 10.1016/0022-2836(89)90527-5. [DOI] [PubMed] [Google Scholar]
  14. Vernet T., Tessier D. C., Chatellier J., Plouffe C., Lee T. S., Thomas D. Y., Storer A. C., Ménard R. Structural and functional roles of asparagine 175 in the cysteine protease papain. J Biol Chem. 1995 Jul 14;270(28):16645–16652. doi: 10.1074/jbc.270.28.16645. [DOI] [PubMed] [Google Scholar]
  15. Walker N. P., Talanian R. V., Brady K. D., Dang L. C., Bump N. J., Ferenz C. R., Franklin S., Ghayur T., Hackett M. C., Hammill L. D. Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell. 1994 Jul 29;78(2):343–352. doi: 10.1016/0092-8674(94)90303-4. [DOI] [PubMed] [Google Scholar]
  16. Wilke M. E., Higaki J. N., Craik C. S., Fletterick R. J. Crystal structure of rat trypsin-S195C at -150 degrees C. Analysis of low activity of recombinant and semisynthetic thiol proteases. J Mol Biol. 1991 Jun 5;219(3):511–523. doi: 10.1016/0022-2836(91)90190-h. [DOI] [PubMed] [Google Scholar]
  17. Willenbrock F., Brocklehurst K. A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and -161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B and of L-arginine 2-naphthylamide catalysed by cathepsin H. Biochem J. 1985 Apr 15;227(2):521–528. doi: 10.1042/bj2270521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wilson K. P., Black J. A., Thomson J. A., Kim E. E., Griffith J. P., Navia M. A., Murcko M. A., Chambers S. P., Aldape R. A., Raybuck S. A. Structure and mechanism of interleukin-1 beta converting enzyme. Nature. 1994 Jul 28;370(6487):270–275. doi: 10.1038/370270a0. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES