Stickiness and Methods for Measurement

In the control experiment, the labeled substrate is in the large solution, and only the enzyme is added in the small volume. The control measures the amount 

Note that A in Eq. (61) is the level of free A in the original incubation, and that if Ef approaches this will be less than the total amount of A added. In practice this is not a serious problem, since the (1 + KJA) term is a correction for not tying up ail of the enzyme as EA, and if is less than , it is easy to add enough A so that almost all of the enzyme is in the EA form. When the binding is loose and one cannot get enough enzyme in solution to convert most of it to EA, this correction term is important, but A will be nearly equal to total A added in this case. 

Note also in Eq. (61) that it is k, and not Vi/Et that is compared to k (ks includes catalysis and release of the first product). It may be that a substrate has a low stickiness ratio and yet is still released more slowly than Vi/ , if ks is much greater than the rate constant for second product release. 

The rate with which A dissociates from EA relative to Vi/ , (this is a comparison with the turnover number) is then given by 

In Eqs. (61) and (62), one must use a value for ATja from other experiments, and this is often the most poorly known kinetic constant. Where it is not possible to tie up a high percentage of the enzyme as EA, one can run the experiments with different levels of A as well as B, and then the equation for the amount of labeled product will be 

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