Direct Structural Observation of an Acyl-Enzyme Intermediate in the Hydrolysis of an Ester Substrate by Elastase

Abstract

The method of X-ray crystallographic cryoenzymology has been used to determine the crystal structure of a kinetically significant species on the reaction pathway of a crystalline enzyme. The structure of a specific acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of the N-carbobenzoxy-l-alanine p-nitrophenyl ester has been determined and refined against X-ray diffraction data at 2.3-Å resolution. The difference Fourier electron density map clearly shows electron density for the trapped acyl-enzyme. The acyl-enzyme was formed at −26 °C and was stabilized at −55 °C during data collection, taking advantage of the glass transition in protein dynamics that occurs at around −50 °C. © 1994, American Chemical Society. All rights reserved.