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 TV-carbobenzoxy-L-alanine p-nitrophenylester has been determined and refined against X-ray diffraction data at 2.3-A 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.