Gentamicin acetyltransferase I will catalyze acyl transfer from chloroacetylcoenzyme A to form 3-N-chloroacetylgentamicin. This product can be linked to coenzyme A to form a multisubstrate analog by nucleophilic displacement of the chlorine by the sulfur of coenzyme A. The analog can be purified by selective binding to cationic and anionic ion exchange resins. Kinetic analysis of a time-dependent onset and reversal of inhibition of gentamicin acetyltransferase I by the purified multisubstrate analog yields an inhibition constant of 5 approximately 20 x 10(-10) M. The inhibitor does not potentiate antibiotic activity against resistant Escherichia coli. Nevertheless, the effectiveness of the tight-binding between the enzyme and the multisubstrate analog demonstrates that inhibitors of resistance can be designed and prepared by specific enzymatic synthesis.