Abstract
Abstract
Truncating an accessory fragment from the substrate or substituting an active-site side chain in the enzyme catalyst generally results in a decrease in kcat/Km for the catalyzed reaction, and the activity lost may sometimes be recovered in third-order reactions of the substrate or enzyme fragments. The effective molarity (EM) for a fragment truncated from the substrate or for a substituted active-site side chain at the transition state for the whole enzyme-catalyzed reaction is equal to kcat/Km (M–1 s–1) for the wild-type enzyme-catalyzed reaction divided by the rate constant (M–2 s–1) for the third-order enzyme-catalyzed reaction of the substrate or enzyme pieces. These EMs are the predicted concentration of the deleted substrate or enzyme piece in the third-order reaction required to give the reaction velocity observed for the wild-type enzyme-catalyzed reaction. The EMs determined for a variety of enzymatic reactions range from 4 to 109 M. These variations are interpreted using a model that assumes an ≈6 kcal/mol advantage for the reaction of whole species that arises from the larger entropic barrier to the higher-order reactions of enzyme or substrate fragments. Interactions that specifically stabilize or destabilize the transition states for enzyme-catalyzed reactions of the fragments also affect the EM. Large EMs are observed when fragment binding to the enzyme is impaired, and small EMs result when the fragment complex is stabilized by interactions between second-shell active-site side chain(s). The reported EM values are largely entropically controlled, consistent with a high degree of structural organization at the active sites for the wild-type and variant enzymes.