![]() ![]() 1,4 Other examples include the processes that maintain constant levels of neurotransmitters, rapidly terminate neural signals, maintain stable folate levels even under conditions of folate deprivation, and prevent de novo DNA methylation during cell replication. Perhaps the most common example of SI comes from housekeeping metabolism, where high levels of ATP suppress glycolysis (and thus halt ATP production) by inhibiting phosphofructokinase. However, multiple physiological phenomena are controlled by SI. SI is often claimed to be an artefact caused by the use of artificially high substrate concentrations in enzymatic assays. 1–3 Instead of reaching a steady-state equilibrium at the maximum reaction speed, the excess substrate starts inhibiting the enzyme-catalyzed reaction. Introduction Substrate inhibition (SI) is a common phenomenon affecting about 25% of known enzymes. ![]() ![]() These results show that substrate inhibition can be caused by substrate binding to the enzyme–product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme–product complex. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. It is generally attributed to the formation of an unproductive enzyme–substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Substrate inhibition is the most common deviation from Michaelis–Menten kinetics, occurring in approximately 25% of known enzymes. ![]()
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