Determination of the transition state structures and mechanisms of enzymatic and nonenzymatic reactions through kinetic isotope effects

Kinetic isotope effects have been measured for isomerization and cleavage reactions of uridine 3'-m-nitrobenzyl phosphate at 86°C and at pH 2.5, 5.5 and 10.5. At pH 2.5 both cleavage and isomerization reactions proceed through a neutral phosphorane intermediate. The kinetic isotope effects for the pH independent isomerization reaction support a stepwise mechanism with a monoanionic phosphorane intermediate. The pH independent cleavage reaction is either a stepwise reaction with a monoanionic phosphorane intermediate or an ANDN reaction with a SN2 transition state. A water mediated proton transfer is absolutely required for the formation of a phosphorane intermediate. The isotope effects measured for the hydroxide catalyzed cleavage reaction are consistent with a concerted reaction with a transition state in which the leaving group departs with almost a full negative charge.;The regiospecificity of the reaction catalyzed by kanamycin nucleotidyltransferase of kanamycin A with either ATP or m-nitrobenzyl triphosphate, a slow substrate analogue, was determined directly by one- and two-dimensional hetero- and homonuclear NMR techniques. The kinetic isotope effects measured are consistent with a concerted reaction with a slightly associative transition state structure for the reaction catalyzed by kanamycin nucleotidyltransferase.;In the phosphodiesterase reaction with adenosine-5' p-nitrophenyl phosphate the 15(V/K) isotope effect is unity which may either indicate that the chemistry is not rate limiting or that the leaving group is being protonated as it departs. A normal secondary 18O isotope effect was measured for the adenosine-5' p-nitrophenyl phosphate reaction. In the absence of the primary isotope effects it is not possible to make any definitive conclusion on the structure of the transition states for these reactions.;In light of the similarity between UDP-galactose 4-epimerase and dTDP-glucose 4,6-dehydratase, the triad Thr134, Tyr160 and Lys164 has been shown by mutagenesis and kinetic studies to be important for catalysis of the first hydride transfer in the dTDP-glucose 4,6-dehydratase mechanism. Tyr160 shows an unusually low pKa value in dTDP-glucose 4,6-dehydratase and it is believed to act as the base catalyst in the first hydride transfer step. Lys 164 and Thr134 play an important role in the first step by stabilizing the oxyanion of Tyr160.