Kinetic mechanism of kanamycin nucleotidyltransferase from Staphylococcus aureus

Kanamycin nucleotidyltransferase (KNTase) catalyzes the transfer of the ade nyl group from MgATP to either the 4' or 4 "-hydroxyl group of aminoglycosi de antibiotics. The steady state kinetic parameters of the enzymatic reacti on have been measured by initial velocity, product, and dead-end inhibition techniques. The kinetic mechanism is ordered where the antibiotic binds pr ior to MgATP and the modified antibiotic is the last product to be released . The effects of altering the relative solvent viscosity are consistent wit h the release of the products as the rare-limiting step. The pH profiles fo r 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 proton ated 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 am inoglycoside antibiotics and 5 nucleotide triphosphates indicates very litt le difference in the rate of catalysis or substrate binding among these sub strates. (C) 1999 Academic Press.