The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.