Kinetic mechanism of kanamycin nucleotidyltransferase from Staphylococcus aureus

Kanamycin nucleotidyltransferase (KNTase) catalyzes the transfer of the adenyl group from MgATP to either the 4' or 4 "-hydroxyl group of aminoglycoside antibiotics. The steady state kinetic parameters of the enzymatic reaction have been measured by initial velocity, product, and dead-end inhibition techniques. The kinetic mechanism is ordered where the antibiotic binds prior to MgATP and the modified antibiotic is the last product to be released. The effects of altering the relative solvent viscosity are consistent with the release of the products as the rare-limiting step. The pH profiles for V-max and V/K-ATP show that a single ionizable group with a pK of similar to 8.9 must be protonated for catalysis. The V/K profile for kanamycin as a function of pH is bell-shaped and indicates that one group must be protonated with a pK value of 8.5, while another group must be unprotonated with a pK value of 6.6. An analysis of the kinetic constants for 10 different aminoglycoside antibiotics and 5 nucleotide triphosphates indicates very little difference in the rate of catalysis or substrate binding among these substrates. (C) 1999 Academic Press.