Characterization of the transition-state structure of the reaction of kanamycin nucleotidyltransferase by heavy-atom kinetic isotope effects.
The transition-state structure for the reaction catalyzed by kanamycin nucleotidyltransferase has been determined from kinetic isotope effects. The primary (18)O isotope effects at pH 5.7 (close to the optimum pH) and at pH 7.7 (away from the optimum pH) are respectively 1.016 +/- 0.003 and 1.014 +/- 0.002. Secondary (18)O isotope effects of 1.0033 +/- 0.0004 and 1.0024 +/- 0.0002 for both nonbridge oxygen atoms were measured respectively at pH 5.7 and 7.7. These isotope effects are consistent with a concerted reaction with a slightly associative transition-state structure.
Gerratana
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Institute for Enzyme Research, Department of Biochemistry, College of Agricultural Life Sciences, 1710 University Avenue, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Frey
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PA
Cleland
W W
WW
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D007612:Kanamycin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D010103:Oxygen Isotopes / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D000249:Adenosine Monophosphate; D007612:Kanamycin; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase