title: Imprinting of lyophilized alpha-chymotrypsin affects the reactivity of the active-site imidazole.
abstract: Iodomethane reacted in vacuo with lyophilized alpha-chymotrypsin to give an inactive enzyme in which the active-site imidazole was dimethylated. However, alpha-chymotrypsin co-lyophilized with the competitive inhibitors, N-acetyl-L-tryptophan or N-acetyl-D-tryptophan, was fully protected from such inactivation. In contrast, indole by itself not only did not protect the lyophilized enzyme from inactivation by iodomethane but also increased the rate of inactivation. The lyoprotectants citrate or sorbitol also showed opposite effects when co-lyophilized with alpha-chymotrypsin. Citrate protected the lyophilized enzyme from inactivation, while bound sorbitol dramatically accelerated the inactivation. Imprinting of lyophilized alpha-chymotrypsin with indole or sorbitol increased the reactivity of the active-site histidine towards iodomethane. Co-lyophilization of alpha-chymotrypsin with appropriate ligands is known to increase significantly its enzymatic activity in hydrophobic organic solvents. It is proposed that this imprinting phenomenon arises because a greater proportion of the active-sites in the lyophilized enzyme are in a catalytically favorable conformation where the imidazole of His-57 is more strongly hydrogen bonded to the carboxylate of Asp-102.
authors.affiliation: Department of Chemistry, University of Ottawa, Ontario, Canada.
authors.forename: N A
authors.initials: NA
authors.lastname: Stewart
authors.forename: A
authors.initials: A
authors.lastname: Taralp
authors.forename: H
authors.initials: H
authors.lastname: Kaplan
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D004789:Enzyme Activation; D004795:Enzyme Stability; D006639:Histidine / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D014618:Vacuum
chemical_list: D007093:Imidazoles; D008024:Ligands; D006639:Histidine; C029899:imidazole; D002918:Chymotrypsin