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X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate

Nature Structural Biology volume 8, pages 689–694 (2001)Cite this article

Abstract

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl–enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme–product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the β-sheet of the active site.

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Figure 1: Hydrolsysis of the acyl–enzyme intermediate formed between β-casomorphin-7 (YPFVEPI) and porcine pancreatic elastase.
Figure 2: Refined 2mFo − DFc electron density for the acyl–enzyme intermediate and the assigned tetrahedral intermediate.
Figure 3: Isotropic temperature factors in the acyl–enzyme complex and in the tetrahedral intermediate (b) formed between porcine pancreatic elastase and human β-casomorphin-7.
Figure 4: Structural changes within the peptide binding pocket during catalysis.

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Acknowledgements

We gratefully acknowledge the assistance of W. Burmeister, S. Wakatsuki, R. Wouts and A. Kirrander during data collection. Work was supported by the B.B.S.R.C., E.P.S.R.C., M.R.C., the Swedish Research Council, NFR, the EU BIOTECH programme, AstraZeneca Pharmaceuticals (via a CASE award to P.A.W.) and New College, Oxford (via a Junior Research Fellowship to R.C.W.). We thank ESRF and the EMBL Outstation in Grenoble for beam time and support.

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Author notes

  1. Richard Neutze

    Present address: Department of Molecular Biotechnology, Chalmers University of Technology, P.O. Box 462, SE 40530, Göteborg, Sweden

  2. Rupert C. Wilmouth, Karl Edman, Thomas R. Schneider and Janos Hajdu: These authors contributed equally to this paper.

Authors and Affiliations

  1. The Dyson Perrins Laboratory and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QY, UK

    Rupert C. Wilmouth, Penny A. Wright, Ian J. Clifton & Christopher J. Schofield

  2. Department of Biochemistry, Uppsala University, Box 576, Biomedical Centre, Uppsala, SE-75123, Sweden

    Karl Edman & Richard Neutze

  3. Department of Structural Chemistry, University of Göttingen, Tammannstrasse 4, Göttingen, 37077, Germany

    Thomas R. Schneider

  4. Janos Hajdu

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Correspondence to Christopher J. Schofield or Janos Hajdu.

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Wilmouth, R., Edman, K., Neutze, R. et al. X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate. Nat Struct Mol Biol 8, 689–694 (2001). https://doi.org/10.1038/90401

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