Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(g), calculated from K-m values) and the activation energy (DeltaG(T), determined from k(cat)/K-m values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(g) for oligopeptides with 15 amino acid replacements at each of the positions P-1', P-1, and P-2 were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(g). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis. (C) 2002 Elsevier Science.