PUTTING KINETIC PRINCIPLES INTO PRACTICE

The overall goal of efforts to characterize enzymes is to document their molecular and kinetic properties. Regardless of the exact mechanism of an enzyme reaction, a kinetic characterization often makes use of the simple Michaelis-Menten model  

Once these kinetic constants are determined, the specificity constant for various substrates and under defined conditions can be obtained as 

Since significant meaning is placed on these measured constants and parameters, it is important that they be determined accurately and unambiguously. It is also important that the reader or practitioner in the field of enzymology be able to assess if the measurement of these parameters is reliable. Furthermore, since enzyme behavior is often modeled as Michaelis-Menten (hyperbolic) kinetics, it seems reasonable that interpretations of observations should be made in the context of the Michaelis-Menten model. In some cases, alternative explanations for enzyme kinetic behavior may be appropriate and one may be inclined to select one interpretation over another (preferably based on a kinetic analysis, although too often this is done on intuition). 

Perhaps the most elementary consideration that should be satisfied is that the measured rates of enzyme reactions under aU conditions represent initial velocities (vq). The indication that initial rates or linear rates were measured are other ways to convey that this standard of experimentation has been met. One of the original stipulations of the general applicability of the Michaelis-Menten model (as well as many others) is that d[So /dt 0 during the time period over which the rate of product formation is measured. Thus, the measured reaction rate is representative of that taking place initially at the [So] selected. This condition is especially important at low [Sol values, where reaction rates are nearly first order with respect to [Sol- In practice, up to 5 to 10% depletion of [Sol can be tolerated over the time frame used to assay [P] for the purpose of determining reaction rates, because error caused by normal experimental 

Continuous assay procedures facilitate estimation of initial rates since the opportunity exists to linearize the initial portion of the reaction progress curve (Fig. 14.1). In contrast, the fixed-point assay, where the reaction or assay is quenched at a preselected interval(s) to allow for product measurement, requires greater care and vigilance to ensure that an estimation of initial velocity was obtained d ydt must be linear during the entire assay period). Using the data in Fig. 14.1 as an example, a fixed-point assay interval of lO-h minutes would not provide for an estimate of initial velocity, whereas intervals of 6 minutes or less would. 

---