Site-specific mutation of Glu-166 to Ala in beta-lactamase causes a millionfold reduction in catalytic activity toward both penicillin and cephalosporin substrates and results in the stoichiometric accumulation of a normally transient acyl-enzyme intermediate. Kinetic analysis indicated that substitution of Glu-166 by Ala leads to negligible effect on the acylation half of the reaction but effectively eliminates the deacylation reaction. Such differential effects on the rates of formation and breakdown of an enzyme-substrate intermediate have not been previously reported. Thus, unlike the situation for most transfer enzymes, e.g., the serine proteases, acylation and deacylation in beta-lactamase catalysis are not "mirror" images and must involve different mechanisms. The results suggest an explanation for the different catalytic activities between the beta-lactamases and the penicillin-binding proteins involved in bacterial cell-wall synthesis.