Biophys J, July 1999, p. 85-98, Vol. 77, No. 1

Consequences of Breaking the Asp-His Hydrogen Bond of the Catalytic Triad: Effects on the Structure and Dynamics of the Serine Esterase Cutinase

Department of Chemistry, University of California, Santa Barbara, California 93106 USA

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp¹⁷⁵-His¹⁸⁸-Ser¹²⁰ of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp¹⁷⁵-His¹⁸⁸ hydrogen bond intact, and in two simulations the Asp¹⁷⁵-His¹⁸⁸ hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp¹⁷⁵-His¹⁸⁸ hydrogen bond. Short contacts of less than 2.6 Å were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp¹⁷⁵ and the NE2 of His¹⁸⁸. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp¹⁷⁵-His¹⁸⁸ hydrogen bond is broken. In the two simulations in which the Asp¹⁷⁵-His¹⁸⁸ hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp¹⁷⁵ and His¹⁸⁸ are more than 6 Å apart in the final structure from dynamics, and the side chains of Ser¹²⁰ and Asp¹⁷⁵ are in closer proximity to the NE2 of His¹⁸⁸ than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp¹⁷⁵-His¹⁸⁸ hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser⁴² and Gln¹²¹, is stable. Only one of the hydrogen bonds (Ser⁴² OG to Gln¹²¹ NE2) observed in the crystal structure that stabilize the conformation of Ser⁴² OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.

Biophys J, July 1999, p. 85-98, Vol. 77, No. 1
© 1999 by the Biophysical Society   0006-3495/99/07/85/14  $2.00

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