Permeable membrane/mass spectrometric measurement of an enzymatic kinetic isotope effect: .alpha.-chymotrypsin-catalyzed transesterification

• Published in: Journal of the American Chemical Society Vol. 105; no. 23; pp. 6935 - 6941
• Main Authors: Calvo, K. C, Weisenberger, C. R, Anderson, L. B, Klapper, Michael H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.11.1983
• Subjects: alpha -chymotrypsin; Analytical, structural and metabolic biochemistry; Biological and medical sciences; deacylation; Enzymes and enzyme inhibitors; ethyl 2-furoic acid; ethyl 5-n-propyl-2-furoic acid; Fundamental and applied biological sciences. Psychology; Hydrolases; mass spectroscopy; Reaction mechanism; Chymotrypsin A; Isotope effect; Enzyme
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja00361a032

The super(16)O/ super(18)O kinetic isotope effect (KIE) associated with an alpha -chymotrypsin-catalyzed transesterification was measured by using the technique of permeable membrane/mass spectroscopy. This almost in situ method is bsed upon the ability of the ester product, but not water and other polar compounds, to permeate through a dimethyl silicone membrane, which separates the aqueous reaction solution from the evacuated inlet of the mass spectrometer. The time course of both super(16)O and super(18)O product formation can be followed simultaneously, permitting rapid KIE measurement. With super(18)O-substituted ethanol, the KIE for the formation of both ethyl 2-furoate and ethyl 5-n-propyl-2-furoate from the respective p-nitrophenyl esters is initially normal and decreases with time to reach within approximately 2 min at 25 degree C, pH 8.5, a constant value of 1.009 plus or minus 0.007 for the former and 0.90 plus or minus 0.02 for the latter. The large inverse steady-state KIE associated with the formation of ethyl 5-n-propyl-2-furoate decreases when the temperature is raised or when the pH is lowered. The pH dependence can be fit with an apparent pK sub( alpha ) of 7.3. The super(16)O/ super(18)O KIE (ethyl ester oxygen) for the nonenzymatic, alkaline hydrolysis of ethyl 2-fuorate was measured as 1.012 plus or minus 0.010 at 25 degree C.