Kinetic Mechanism from the Variation of Substrates and Products

5 KINETIC MECHANISM FROM THE VARIATION OF SUBSTRATES AND PRODUCTS 

Kinetic isotope effects are sensitive to changing concentrations of substrates, products of reactions, and allosteric effectors (Cook, 1991). This sensitivity of isotope effects may be profitably applied to analyze luetic mechanisms. Consider the following reaction scheme for a bisubstrate reaction  

The kinetic isotope effects in bisubstrate reactions are measured by placing an isotope label in one of the substrates, followed by a complete kinetic analysis with a pair of unlabeled substrates, and then by a complete analysis with a pair of labeled and unlabeled substrate. In this way, the kinetic isotope effect on the maximal velocity, V, and the isotope effects on WK for both substrates, (V/lfA) and (V/JRTb), are obtained. Table 1 gives typical examples from the literature. 

Generally, the following can be stated quahtatively concerning the dependence of isotope effects on V and V/K. 

In a sequential mechanism, an isotope effect equal to or close to one on one of the two substrate V/K values suggests a Steady-State Ordered mechanism. The V/K for the first substrate bound will have the isotope effect of unity. The isotope effect of one may also apply to the second substrate in a Ping Pong mechanism, but a distinctive, initial velocity pattern with parallel lines is obtained in this case. 

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