Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase.
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-A resolution. The difference Fourier electron density map clearly shows electron density for the trapped acyl-enzyme. The acyl-enzyme was formed at -26 degrees C and was stabilized at -55 degrees C during data collection, taking advantage of the glass transition in protein dynamics that occurs at around -50 degrees C.
Ding
X
X
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Rasmussen
B F
BF
Petsko
G A
GA
Ringe
D
D
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D004952:Esters; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine; D014961:X-Ray Diffraction / Q000379:methods
D004952:Esters; C088727:N-carbobenzoxyalanine 4-nitrophenyl ester; D010196:Pancreatic Elastase; D000409:Alanine