Location of Repository

Oxyanion and tetrahedral intermediate stabilization by subtilisin : detection of a new tetrahedral adduct

By Nicole Howe, Louis Rogers, Chandralal Hewage and J.Paul G. Malthouse


The peptide-derived glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal has been shown to be ~10 fold more effective as an inhibitor of subtilisin than Z-Ala-Pro-Phe-glyoxal. Signals at 107.2 p.p.m. and 200.5 p.p.m. are observed for the glyoxal keto and aldehyde carbons of the inhibitor bound to subtilisin, showing that the glyoxal keto and aldehyde carbons are sp3 and sp2 hybridized respectively. The signal at 107.2 p.p.m. from the carbon atom attached to the hemiketal oxyanion is formed in a slow exchange process that involves the dehydration of the glyoxal aldehyde carbon. Two additional signals are observed one at 108.2 p.p.m. and the other at 90.9 p.p.m. for the glyoxal keto and aldehyde carbons respectively at pHs 6-8 demonstrating that subtilisin forms an additional tetrahedral adduct with Z-Ala-Ala-Phe-glyoxal in which both the glyoxal keto and aldehyde carbons are sp3 hybridised. For the first time we can quantify oxyanion stabilisation in subtilisin. We conclude that oxyanion stabilisation is more effective in subtilisin than in chymotrypsin. Using 1H-NMR we show that the binding of Z-Ala-Ala-Phe-glyoxal to subtilisin raises the pKa of the imidazolium ion of the active site histidine residue promoting oxyanion stabilisation. The mechanistic significance of these results are discussed

Topics: Subtilisin, Glyoxal inhibitor, Protease, Chymotrypsin, pH, Oxyanion, Proteolytic enzymes, Chymotrypsin, Hydrogen-ion concentration, Subtilisins
Publisher: Elsevier
Year: 2009
DOI identifier: 10.1016/j.bbapap.2009.04.007
OAI identifier: oai:researchrepository.ucd.ie:10197/3022
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://hdl.handle.net/10197/30... (external link)
  • http://creativecommons.org/lic... (external link)
  • Suggested articles



    1. (1989). 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.,
    2. (2002). A 13C-NMR study of the inhibition of delta-chymotrypsin by a tripeptide-glyoxal inhibitor, doi
    3. (1997). A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct,
    4. (1996). A low-barrier hydrogen bond in subtilisin: 1H and 15N NMR studies with peptidyl trifluoromethyl ketones, doi
    5. (1990). A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain, doi
    6. (1992). A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin,
    7. (1995). A study of the stabilization of the oxyanion of tetrahedral adducts by trypsin, chymotrypsin and subtilisin, doi
    8. (1960). Acid ionization Constants of Alcohols.
    9. (2007). An NMR study of the inhibition of pepsin by glyoxal inhibitors: Mechanism of tetrahedral intermediate stabilization by the aspartyl proteinases, doi
    10. (1972). An X-Ray Crystallographic Study of the Binding of Peptide Chloromethyl Ketone Inhibitors to Subtilisin BPN, doi
    11. and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes, doi
    12. (2002). Characterisation of a low barrier hydrogen bond in the active site of chymotrypsin, doi
    13. (1987). Complex of a-Chymotrypsin and N-Acetyl-L-leucyl-Lphenylalanyl Trifluoromethyl Ketone: doi
    14. (1991). Contribution of the glutamine 19 side chain to transitionstate stabilization in the oxyanion hole of papain, doi
    15. (1978). Correlation analysis in Chemistry, doi
    16. (1998). Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin, doi
    17. (2000). Crystal structure of δ-chymotrypsin bound to a peptidyl chloromethyl ketone inhibitor, Acta Cryst. doi
    18. (1983). Detection of a Tetrahedral Adduct in a Trypsin-Chloromethyl Ketone Specific Inhibitor Complex by 13C NMR, doi
    19. (1996). Determination of the ionization state of the activesite histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR,
    20. (2007). Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors, doi
    21. (2007). Developments in the characterisation of the catalytic triad of alpha-chymotrypsin: Effect of the protonation state of doi
    22. (1979). Do cleavages of amides by serine proteases occur through a stepwise pathway involving tetrahedral intermediates?, doi
    23. (1995). Exceptional active site H-bonding in enzymes? Significance of the 'oxyanion hole' in the serine proteases from a model study, doi
    24. (1989). How Do Serine Proteases Really Work?, doi
    25. (1999). Hydrogen Bonding to Active-Site Histidine in Peptidyl Boronic Acid Inhibitor Complexes of Chymotrypsin and Subtilisin: Proton Magnetic Resonance Assignments and H/D Fractionation, doi
    26. (1998). Improved WATERGATE Pulse Sequences for Solvent Suppression in doi
    27. (2005). Ionisations within a subtilisinglyoxal inhibitor complex, doi
    28. (1956). Kinetics of the hydration of acetaldehyde, doi
    29. (1995). Modification of the electrostatic environment is tolerated in the oxyanion hole of the cysteine protease papain, doi
    30. (1992). Oxyanion Hole Interactions in Serine and Cysteine Proteases, doi
    31. (1981). pKa Prediction for Organic Acids and Bases, Chapman and Hall, London and doi
    32. (2008). Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases, doi
    33. (1973). Specificity of chymotrypsin. Separation of Polar, Steric, and Specific Effects in the α-chymotrypsin-catalysed Hydrolysis of AcylSubstituted p-Nitrophenyl esters, doi
    34. (1976). Structure and Mechanism of Chymotrypsin, doi
    35. (1970). Structure of Crystalline a-Chymotrypsin, IV. The Structure of Indoleacryloyl-α-Chymotrypsin and its Relevance to the Hydrolytic Mechanism of the Enzyme,
    36. (1972). Subtilisin; a Stereochemical Mechanism Involving Transition-State Stabilization, doi
    37. (1982). Tar, Tetrahedral Intermediate in Acyl transfer Reactions. A Revaluation of the Significance of Rate Data Used in Deriving Fundamental Linear free Energy Relationships, doi
    38. (2007). The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases, doi

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.