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J. Biol. Chem., Vol. 280, Issue 27, 25644-25650, July 8, 2005

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Energetic and Structural Consequences of Perturbing Gly-193 in the Oxyanion Hole of Serine Proteases^*

Kevin M. Bobofchak, Agustin O. Pineda, F. Scott Mathews, and Enrico Di Cera

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

The oxyanion hole of serine proteases is formed by the backbone N atoms of the catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1 residue of substrate in an important H-bonding interaction. The energetic contribution of this interaction in the ground and transition states is presently unknown. Measurements of the individual rate constants defining the catalytic mechanism of substrate hydrolysis for wild-type thrombin and trypsin and their G193A and G193P mutants reveal that Gly-193 is required for optimal substrate binding and acylation. Crystal structures of the G193A and G193P mutants of thrombin bound to the active site inhibitor H-D-Phe-Pro-Arg-CH₂Cl document the extent of perturbation induced by the replacement of Gly-193. The Ala mutant weakens the H-bonding interaction of the N atom of residue 193, whereas the Pro substitution abrogates it altogether with additional small shifts of the protein backbone. From the kinetic and structural data, we estimate that the H-bonding interaction in the oxyanion hole contributes a stabilization of the ground and transition states of >1.5 kcal/mol but <3.0 kcal/mol. These results shed light on a basic aspect of the enzyme-substrate interaction in the entire family of trypsin-like serine proteases.

Received for publication, March 30, 2005 , and in revised form, May 6, 2005.

^* This work was supported by National Institutes of Health Research Grants HL49413, HL58141, and HL73813. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1Z8I and 1Z8J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail: enrico{at}wustl.edu.

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