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Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases

TitleStructural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases
Publication TypeJournal Article
Year of Publication2004
AuthorsGloster, TM, Macdonald, JM, Tarling, CA, Stick, RV, Withers, SG, Davies, GJ
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume279
Pagination49236-49242
Date PublishedNOV 19
ISSN0021-9258
Abstract

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an ``inhibition profile.{''} Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display K(i) values around 500 nM. X-ray structures with both a beta-glucosidase, at 2 Angstrom resolution, and an endoglucanase at atomic (similar to1Angstrom) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of k(cat)/K(m) and 1/K(i) with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/K(i) in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/K(i) and the pH dependence of k(cat)/K(m), the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.

DOI10.1074/jbc.M407195200