The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.