Effects of chemical modulators on enzyme specificity

Chemical inhibitors bind to enzymes, thereby inhibiting their catalytic activity. While many enzymes catalyze reactions with a single substrate, others, like DNA polymerase, can act on multiple related substrates. Substrate-selective inhibitors (SSIs) target these multi-substrate enzymes to modulate their specificity. Although SSIs hold promise as therapeutics, our theoretical understanding of how different inhibitors influence enzyme specificity remains limited. In this study, we examine enzyme selectivity within kinetic networks corresponding to known inhibition mechanisms. We demonstrate that competitive and uncompetitive inhibitors do not affect substrate specificity, regardless of rate constants. In contrast, noncompetitive and mixed inhibition can alter specificity and can lead to non-monotonic responses to the inhibitor. We show that mixed and non-competitive inhibitors achieve substrate-selective inhibition by altering the effective free-energy barriers of product formation pathways that are enabled by the inhibitors presence. We then apply this framework to the Sirtuin-family deacylase SIRT2, showing that the suicide inhibitor thiomyristoyl lysine (TM) cannot influence substrate specificity unless there is a direct substrate exchange reaction or biochemical constraints are relaxed. These findings provide insights into engineering systems where cofactor binding modulates metabolic flux ratios.