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 primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent kinase, APH(3')-IIIa, which phosphorylates such antibiotics as kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic 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 (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with 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 addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.
McKay
G A
GA
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Wright
G D
GD
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002384:Catalysis; D004352:Drug Resistance, Microbial; D016983:Enterococcus / Q000201:enzymology*; D007554:Isotopes; D019868:Kanamycin Kinase; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics; D014783:Viscosity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D007554:Isotopes; D012997:Solvents; D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D000251:Adenosine Triphosphatases