Acylenzyme mechanism and solvent isotope effects for cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate.
The mechanism of cholesterol esterase- (carboxylic ester hydrolase, EC 8.1.1.1) catalyzed hydrolysis of the water-soluble ester p-nitrophenyl butyrate has been characterized for commercially available preparations from bovine and porcine pancreas and for a purified preparation from porcine pancreas. Kinetic evidence for an acylenzyme mechanism is provided by experiments wherein the butyryl enzyme is trapped by MeOH, EtOH or n-BuOH. For the last alcohol the transacylation product n-butyl n-butyrate was characterized by GC-mass spectrometry. Solvent isotope effects have been measured for Vmax/Km, which is the rate constant for acylation, and for Vmax, which monitors rate-determining deacylation. Isotope effects of 1.5-3 on these rate constants indicate that both steps of the acylenzyme mechanism for cholesterol esterase catalysis involve transition states that are stabilized by general acid-base proton bridges.
D000438:Alcohols / Q000494:pharmacology; D000818:Animals; D002087:Butyrates; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002417:Cattle; D003903:Deuterium / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010654:Phenylbutyrates / Q000378:metabolism*; D012997:Solvents; D002787:Sterol Esterase / Q000378:metabolism*; D013552:Swine
D000438:Alcohols; D002087:Butyrates; D010654:Phenylbutyrates; D012997:Solvents; C033592:4-nitrophenyl butyrate; D003903:Deuterium; D002265:Carboxylic Ester Hydrolases; D002787:Sterol Esterase