Evidence that a ‘dynamic knockout’ in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis

The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, following conflicting conclusions from studies of the N23PP/S148A variant of the Escherichia coli enzyme. By investigating the temperature dependence of kinetic isotope effects, we present evidence that the reduction in the hydride transfer rate constants in this variant is not a direct result of impairment of conformational fluctuations. Instead, the conformational state of the enzyme immediately before hydride transfer, which determines the electrostatic environment of the active site, affects the rate constant for the reaction. Although protein motions are clearly important for binding and release of substrates and products, there appears to be no detectable dynamic coupling of protein motions to the hydride transfer step itself

An excess of catalytically required motions inhibits the scavenger decapping enzyme

The scavenger decapping enzyme hydrolyses the protecting 5′ cap structure from short mRNAs that result from exosomal degradation. Based on static crystal structures and NMR data it is apparent that the dimeric enzyme has to undergo large structural changes to bind substrate in a catalytically competent conformation. Here, we study the yeast enzyme and show that the associated opening-closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by binding of a second ligand to the enzyme, which occurs under high substrate concentrations. We designed a mutant that disrupts the allosteric pathway that links the second binding event to the dynamics and show that this mutant enzyme is hyperactive. Our data reveals a unique mechanism of substrate inhibition, where motions that are required for catalytic activity also inhibit efficient turnover, when they are present in excess