Aminoglycoside 3′-phosphotransferases [APH(3′)s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3′-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3′) types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4′-deoxy-4′,4′-difluorokanamycin A and 4′-deoxy-4′,4′-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3′)-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3′)s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.
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