A test for measuring the effects of enzyme inactivation

In the single-enzyme, single-substrate reaction with non-mechanism-based enzyme inactivation, the formation of the product and inactivation of the enzyme occur independently. For this reaction, we show that the steady-state hypothesis is applicable even when degradation of the enzyme occurs. An equation for the rate of product formation has been derived and it shows Michaelis-Menten kinetics with an apparent Michaelis-Menten constant KMapp = KM + Kδ where Kδ is the enzyme inactivation constant. Use of a Lineweaver-Burk plot yields values for KMapp, which can be used to estimate Kδ and, consequently, the degree of enzyme inactivation in a particular experiment. We employ this methodology to estimate the inactivation constant for the arsenate reductase catalyzed production of arsenite with appreciable enzyme inactivation.