Product dissociation rates assay

The rate-limiting step for the hydrolysis of single-stranded DNA by SNase was subsequently shown to be product dissociation, and substrate binding is diffusion-controlled when the pH is >7.3.219 The pH optimum is between 8.6 and 10.3, and the enzyme is routinely assayed at pH 9.5. Thus, under these conditions, kinetic studies cannot reveal the effect of mutations on the chemical step. On the basis of the dependence of rate on pH it was proposed that Glu-43 does not act as a general base.219 It was also proposed that residues in a protein loop near the active site may be involved in the physical process that is the rate-determining step.219... 

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