CATALYTIC MECHANISM OF ENTEROCOCCAL KANAMYCIN KINASE (APH(3')-IIIA) -VISCOSITY, THIO, AND SOLVENT ISOTOPE EFFECTS SUPPORT A THEORELL-CHANCE MECHANISM

Bacterial resistance to the aminoglycoside antibiotics is manifested p rimarily through the production of enzymes which covalently modify the se drugs. The Enterococci and Staphylococci produce an ATP-dependent k inase, APH(3')-IIIa, which phosphorylates such antibiotics as kanamyci n, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kin etic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G . D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3 ')-IIIa exhibits small solvent (V-H/V-D approximate to 1.50) and thio effects (V-ATP/V-ATP gamma S = 2) indicating hydroxyl group deprotonat ion and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined wit h the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In add ition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformationa l change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.