A general method for the analysis of random bisubstrate enzyme mechanisms

Abstract

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.

Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle

“…The first enzyme catalyses the oxidation of β-D-glucose to D-glucono-δ-lactone with a production of hydrogen peroxide (H 2 O 2 ). This is a well characterized reaction 24,25 , used herein as a model. Then, hydrogen peroxide serves as a substrate to HRP, which oxidizes Amplex Red (AR; N-acetyl-3,7-dihydroxyphenoxazine), to form the highly fluorescent species resorufin 26 (Figure 1).…”