N-15 isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: Evidence for a tetrahedral intermediate in the mechanism

N-15 isotope effects have been measured on the hydrolysis of glutamine cata lyzed by carbamyl phosphate synthetase of Escherichia coli, The isotope eff ect in the amide nitrogen of glutamine is 1.0217 at 37 degrees C with the w ild-type enzyme in the presence of MgATP and HCO3- (overall reaction taking place), This V/K isotope effect indicates that breakdown of the tetrahedra l intermediate formed with Cys 269 to release ammonia is the rate-limiting step in the hydrolysis. A full isotope effect of 1.0215 is also seen in the partial reaction catalyzed by an E841K mutant enzyme, whose rate of glutam ine hydrolysis is not affected by MgATP and HCO3-. With wild-type enzyme in the absence of MgATP and HCO3-, however, the N-15 isotope effect is reduce d to 1.0157. These isotope effects are interpreted in terms of partitioning of the tetrahedral intermediate whose rate of formation is dependent upon a conformation change which closes the active site after glutamine binding and prepares the enzyme for catalysis. An Ordered Uni Bi mechanism for glut amine hydrolysis that is consistent with the isotope effects and with the c atalytic properties of the enzyme is proposed.