Kinetics and mechanism for enzyme-catalyzed reactions of substrate pieces.

The most important difference between enzyme and small molecule catalysts is that only enzymes utilize the large intrinsic binding energies of nonreacting portions of the substrate in stabilization of the transition state for the catalyzed reaction. A general protocol is described to determine the intrinsic phosphodianion binding energy for enzymatic catalysis of reactions of phosphate monoester substrates, and the intrinsic phosphite dianion binding energy in activation of enzymes for catalysis of phosphodianion truncated substrates, from the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates. The enzyme-catalyzed reactions so-far documented that utilize dianion binding interactions for enzyme activation; and, their phosphodianion truncated substrates are summarized. A model for the utilization of dianion binding interactions for enzyme activation is described. The methods for the determination of the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates, from initial velocity data, are described and illustrated by graphical plots of kinetic data. The results of studies on the effect of site-directed amino acid substitutions at orotidine 5'-monophosphate decarboxylase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase provide strong support for the proposal that these enzymes utilize binding interactions with the substrate phosphodianion to hold the protein catalysts in reactive closed conformations.