A general method for the analysis of random bisubstrate enzyme mechanisms.
In the present communication, a general method for the kinetic analysis of random bisubstrate mechanisms is described. The method comprises a stepwise application of the following kinetic and ligand-binding experiments: determination of steady-state kinetic constants, product inhibition patterns, maximum rate relationships, application of alternate substrates, application of dead-end inhibitors, direct binding of substrates, kinetic isotope effects, and isotope exchange studies. This general method was applied to a practical example: a yeast alcohol dehydrogenase-catalyzed oxidation of 2-propanol by NAD(+) at pH 7.0, 25 degrees C. It was found that this fully reversible reaction proceeds by a steady-state random Bi-Bi mechanism, whereby both dead-end complexes are formed.
Leskovac
Vladimir
V
Faculty of Technology, Bulevar Cara Lazara 1, 21000, Novi Sad, Yugoslavia. leskovac@neobee.net
Trivić
Svetlana
S
Pericin
Draginja
D
Kandrac
Julijan
J
D019840:2-Propanol / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D003903:Deuterium; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D017711:Nonlinear Dynamics; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology*
D009243:NAD; D003903:Deuterium; D000426:Alcohol Dehydrogenase; D019840:2-Propanol