Determination of Enzyme Catalytic Parameters from the Progress Curve

5 DETERMINATION OF ENZYME CATALYTIC PARAMETERS FROM THE PROGRESS CURVE 

It is theoretically possible to derive Vmax and values for an enzyme from a single progress curve (Fig. 3.11). This is certainly an attractive proposition since measuring initial velocity as a function of several substrate concentrations can be a lengthy and tedious task. The velocity of an enzyme-catalyzed reaction can be determined from the disappearance 

In this model, [Sj] is not an explicit function of time. This can represent a problem since most commercially available curve-fitting programs caimot fit implicit functions to experimental data. Thus, to be able to use this implicit function in the determination of / cat and Km, it is necessary to modify its form and transform the experimental data accordingly. Dividing both sides by t and Km and rearranging results in the expression 

A plot of ln([So]/[S ]) versus [Sq —S,]/t yields a straight line with slope =-l/ T, x-intercept = Vmax, and y-intercept = V ,ax/ m (Fig. 3.11). The values of the slope and intercept can readily be obtained using linear regression. Thus, from a single progress curve (i.e., a single [Sf]-t data set) it is possible to obtain estimates of Km and kcm- 

If this procedure sounds too good to be true, it probably is. The major problem with this procedure is that the following conditions must be met  

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