Kinetic mechanism of kanamycin nucleotidyltransferase from Staphylococcus aureus Academic Article uri icon

abstract

  • 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 ratc- limiting step. The pH profiles for V(max) and V/K(ATP) show that a single ionizable group with a pK of 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.

published proceedings

  • BIOORGANIC CHEMISTRY

author list (cited authors)

  • Chen-Goodspeed, M., Vanhooke, J. L., Holden, H. M., & Raushel, F. M.

citation count

  • 28

complete list of authors

  • Chen-Goodspeed, M||Vanhooke, JL||Holden, HM||Raushel, FM

publication date

  • January 1999