Progress curve analysis of adenosine deaminase-catalyzed reactions.
The kinetic constants of the adenosine deaminase-catalyzed conversion of adenosine to inosine were found to be readily obtainable by analyzing the progress curve of a single reaction. A novel inhibitor, 9-(1-hydroxymethyl-3-methylbutyl)adenine, was studied to test the validity of the progress curve method with this enzyme. Estimates of kinetic constants determined by this method were compared to those determined by the conventional initial velocity analysis. The Km and Vmax values for adenosine and the Ki value for the inhibitor were estimated to be 26.1 microM, 1.27 mumol/min/unit of enzyme, and 0.48 microM, respectively, by the initial velocity method, and 29.3 microM, 1.27 mumol/min/unit of enzyme, and 0.52 microM, respectively, by the progress curve analysis. The inhibitor was shown to act competitively with substrate by both methods of analysis. The progress curve experiments were very simple to perform and the constants were calculated (with an interfaced microcomputer) within a few minutes of the completion of each assay.
D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D007288:Inosine / Q000096:biosynthesis; D007700:Kinetics; D009700:Nucleoside Deaminases / Q000378:metabolism*
D007288:Inosine; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000241:Adenosine