KINETIC STUDIES OF ENZYME MECHANISM

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The purpose of this project is to study the transition state of the reaction of kanamycin nucleotidyltransferase (KNT) using isotope effects. KNT catalyzes the transfer of a nucleotide residue from ATP, GTP or UTP to the 4?-hydroxyl group of kanamycin A, inactivating the aminoglycoside itself. The remote label method will be used to study the transition state. Both primary and secondary effects will be studied. The first ones in order to determine that the rate limiting step is the chemical step of the reaction, the second ones give a measure of the change in bond order in the trasition state in atoms not directly involved in cleavage. Thus, synthesis of labeled ATP molecules and of labeled analog are carried out. m-nitrobenzyl triphosphate is an analog of ATP and it will be used in the isotopic measurements.