The Role of Substrate Unbinding in Michaelis-Menten Enzymatic Reactions

Abstract

The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover - turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase-space, we identify four distinct categories of unbinding: inhibitory, excitatory, super-excitatory and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding, but will also allow isolated determination of the distribution of time required for the completion of a catalytic step embedded within a full enzymatic turnover cycle.