Studies on the Mechanism of Action of Penicillopepsin

  • Theo Hofmann
  • Max Blum
  • Annie Cunningham
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 306)


Several high resolution structures of aspartic proteinases are now known, (see Gilliland et al.1 for a recent list and earlier contributions in this volume) and detailed information has been obtained on the binding of substrate analogue inhibitors from the crystallographic analysis of their complexes with the enzymes. Several proposals for their mechanism of action have been made.2–7 Yet we still do not fully understand how these enzymes function. The crystallographic data are insufficient to rationalize the extensive experimental information available from studies on specificity,8 steady-state and presteady-state kinetics,8 isotope exchange experiments,9,10 transpeptidation reactions11–13 and low-temperature kinetics.14 Thus, we do not know much about the molecular events that are responsible for the so-called “secondary specificity”,8 that is, the very large increases in catalytic efficiency with increasing length of substrates, nor do we understand the structural details of transpeptidation reactions. It is also not clear what the rate-determining steps of the reactions are. In our recent studies we have attempted to throw some more light on these phenomena.


Aspartic Proteinase Peptide Bond Cleavage Porcine Pepsin Scissile Bond Solvent Isotope Effect 
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  1. 1.
    G. L. Gilliland, E. L. Winborne, J. Nachman, and A. Wlodawer, The three-dimensional structure of recombinant chymosin at 2.3 Å resolution, Proteins, Struct. Funct. Genet. 8: 82–101 (1990).CrossRefGoogle Scholar
  2. 2.
    K. Suguna, R. R. Bott, E. A. Padlan, E. Subramanian, S. Sherriff, G. Cohen, and D. R. Davies, Structure and refinement at 1.8 Å resolution of the aspartic proteinase from Rhizopus chinensis, J. Mol. Biol. 196: 877–900 (1987).PubMedCrossRefGoogle Scholar
  3. 3.
    L. H. Pearl, The catalytic mechanism of aspartic proteinases, FEBS Lett. 214: 8–12 (1987).PubMedCrossRefGoogle Scholar
  4. 4.
    L. Polgar, The mechanism of action of aspartic proteases involves “push-pull” catalysis, FEBS Lett. 219: 1–4 (1987).PubMedCrossRefGoogle Scholar
  5. 5.
    G. Fischer, Acyl group transfer-aspartic proteinases, in: “Enzyme Mechanisms,” M. I. Page, and A. Williams, eds. pp. 229–239, Roy. Soc. Chem., London 1987.Google Scholar
  6. 6.
    M. N. G. James, and A. R. Sielecki, Aspartic proteinases and their catalytic pathway, in: “Biological Macromolecules and Assemblies. Vol. 3: Active Sites of Enzymes”, F. A. Jurnak and A. McPherson, eds. pp. 415–482, John Wiley and Sons, New York (1984).Google Scholar
  7. 7.
    T. Hofmann, B. M. Dunn, and A. L. Fink, Mechanism of action of aspartic proteinases, Biochemistry 23: 5253–5256 (1984).Google Scholar
  8. 8.
    J. F. Fruton, The mechanism of action of pepsin and related acid proteinases, Adv. Enzymol. 44: 1–36 (1976).PubMedGoogle Scholar
  9. 9.
    V. K. Antonov, L. M. Ginodman, Y. K. Kapitannikov, T. N. Barshevskaya, A. G. Gurova, and L. D. Rumsh, Mechanism of pepsin catalysis: general base catalysis by the active site carboxylate ion, FEBS Lett. 88: 87–90 (1978).PubMedCrossRefGoogle Scholar
  10. 10.
    V. K. Antonov, L. M. Ginodman, L. D. Rumsh, Y. K. Kapitannikov, T. N. Barshevskaya, L. P. Yavashev, A. G. Gurova, and L. I. Volkova, Studies on the mechanism of action of proteolytic enzymes using heavy oxygen exchange, Eur. J. Biochem. 117: 195–200 (1981).PubMedCrossRefGoogle Scholar
  11. 11.
    T. T. Wang, and T. Hofmann, Acyl and amino intermediates in reactions catalyzed by pig pepsin, Biochem. J. 153: 691–699 (1976).PubMedGoogle Scholar
  12. 12.
    M. S. Silver and S. L. T. James, Enzyme catalysed condensation reactions which initiate rapid peptic cleavage of substrates. 2. Proof of mechanism for three examples, Biochemistry 20: 3183–3189 (1980).CrossRefGoogle Scholar
  13. 13.
    M. Blum, A. Cunningham, M. Bendiner and T. Hofmann, Penicillopepsin,the aspartic proteinase from Penicillium janthinellum: Substrate binding and intermediates in transpeptidation reactions, Biochem. Soc. Trans. 13: 1044–1046 (1985).PubMedGoogle Scholar
  14. 14.
    T. Hofmann and A. L. Fink, Cryoenzymology of penicillopepsin, Biochemistry 23: 5247–5253 (1984).PubMedCrossRefGoogle Scholar
  15. 15.
    T. Hofmann, B. Allen, M. Bendiner, M. Blum, and A. Cunningham, Effect of secondary substrate binding in penicillopepsin: contributions of subsites S3 and S2′ to kcat, Biochemistry 27: 1140–1146 (1988).PubMedCrossRefGoogle Scholar
  16. 16.
    B. Allen, M. Blum, A. Cunningham, G. C. Tu, and T. Hofmann, A ligand-induced, temperature-dependent conformational change in penicillopepsin, evidence from nonlinear Arrhenius plots and from circular dichroism studies, J. Biol. Chem. 265: 5060–5065 (1990).PubMedGoogle Scholar
  17. 17.
    M. N. G. James, A. R. Sielecki, F. Salituro, D. H. Rich, and T. Hofmann, Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin, Proc. Natl. Acad. Sci., U.S.A. 79: 6137–6141 (1982).PubMedCrossRefGoogle Scholar
  18. 18.
    T. L. Blundell, J. Cooper, S. I. Foundling, D. M. Jones, B. Atrash, and M. Szelke, On the rational design of renin inhibitors: x-ray studies of aspartic proteinases complexed with transition-state analogues, Biochemistry 26: 5585–5590 (1987).PubMedCrossRefGoogle Scholar
  19. 19.
    A. Sali, B. Veerapandian, J. B. Cooper, S. I. Foundling, D. J. Hoover, and T. L. Blundell, High-resolution x-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme, EMBO J. 8: 2179–2188 (1989).PubMedGoogle Scholar
  20. 20.
    A. Sali, B. Veerapandian, J. B. Cooper, D. S. Moss, T. Hofmann, and T. L. Blundell, Rigid body movement and conformational differences in aspartic proteinases, Proteins: Struct. Funct. Genet. (submitted).Google Scholar
  21. 21.
    K. B. J. Schowen, Solvent hydrogen isotope effects, in: “Transition states of biochemical processes,” R. D. Gandour and R. L. Schowen, eds., pp. 225–283, Plenum Press, New York (1978).Google Scholar
  22. 22.
    G. E. Clement and S. L. Snyder, The kinetics of the pepsin catalyzed hydrolysis of N-acetyl-L-phenyalanyl-L-tyrosine methyl ester, J. Am. Chem. Soc. 88: 5338–5339 (1966).CrossRefGoogle Scholar
  23. 23.
    T. W. Reid and D. Fahmey, The pepsin catalyzed hydrolysis of sulfite esters, J. Am. Chem. Soc. 89: 5941–5943 (1967).CrossRefGoogle Scholar
  24. 24.
    T. R. Hollands and J. S. Fruton, On the mechanism of pepsin action, Proc. Natl. Acad. Sci., U.S.A. 62: 1116–1120 (1969).PubMedCrossRefGoogle Scholar
  25. 25.
    H. Neumann, Y. Levin, A. Berger and E. Katchalski, Pepsin catalyzed transpeptidation of the amino-transfer type, Biochem. J. 73: 33–41 (1959).PubMedGoogle Scholar
  26. 26.
    M. Takahashi, T. T. Wang, and T. Hofmann, Acyl intermediates in pepsin and penicillopepsin catalyzed reactions, Biochem. Biophys. Res. Commun. 57: 39–46 (1974).PubMedCrossRefGoogle Scholar
  27. 27.
    M. K. Lutek, T. Hofmann, and C. M. Deber, Transpeptidation reactions of porcine pepsin: formation of tetrapeptides from dipeptide substrates, J. Biol. Chem. 263: 8011–8016 (1988).PubMedGoogle Scholar
  28. 28.
    M. Blum, A. Cunningham, H. Pang, and T. Hofmann, Mechanism and pathway of penicillopepsin catalyzed transpeptidation and evidence for non-covalent trapping of amino acid and peptide intermediates, J. Biol. Chem. 266: 9501–9507 (1991).PubMedGoogle Scholar
  29. 29.
    A. Cunningham, M. I. Hofmann, and T. Hofmann, Rate-determining steps in penicillopepsin catalyzed reactions, FEBS Lett. 276: 119–122 (1990).PubMedCrossRefGoogle Scholar
  30. 30.
    T. Hofmann, Penicillopepsin, Meth. Enzymol. 45: 434–452 (1976).PubMedCrossRefGoogle Scholar
  31. 31.
    T. Hofmann and R. S. Hodges, A new chromophoric substrate for penicillopepsin and other fungal aspartic proteinases, Biochem. J. 203: 603–610 (1982).PubMedGoogle Scholar
  32. 32.
    T. Hofmann, R. S. Hodges and M. N. G. James, Effect of pH on the activities of penicillopepsin and Rhizopus pepsin, and a proposal for the productive substrate binding mode in penicillopepsin, Biochemistry 23: 635–643 (1984).PubMedCrossRefGoogle Scholar
  33. 33.
    J. S. Fruton, Fluorescence studies on the active site of proteinases, Mol. Cell. Biol. 32: 105–114 (1980).Google Scholar
  34. 34.
    D. H. Rich and M. S. Bernatowicz, Synthesis of analogues of the carboxyl protease inhibitor pepstatin. Effect of structure in subsite P3 on inhibition of pepsin, J. Med. Chem. 25: 791–795 (1982).PubMedCrossRefGoogle Scholar
  35. 35.
    K. L. Schowen, The proton inventory technique, C.R.C. Crit. Rev. Biochem. 17: 1–44 (1984).CrossRefGoogle Scholar
  36. 36.
    C. Abad-Zapatero, D. N. Neidhart, T. J. Rydel, J. Luly, and J. Erickson, Refined crystal structures of porcine pepsin and inhibitor complexes: evidence for a flexible subdomain, (see earlier chapter in this volume)Google Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Theo Hofmann
    • 1
  • Max Blum
    • 1
  • Annie Cunningham
    • 1
  1. 1.Dept. of BiochemistryUniversity of TorontoTorontoCanada

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