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

Abstract

15N isotope effects have been measured on the hydrolysis of glutamine catalyzed by carbamyl phosphate synthetase of Escherichia coli. The isotope effect in the amide nitrogen of glutamine is 1. 0217 at 37 degrees C with the wild-type enzyme in the presence of MgATP and HCO(3)(-) (overall reaction taking place). This V/K isotope effect indicates that breakdown of the tetrahedral 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 glutamine hydrolysis is not affected by MgATP and HCO(3)(-). With wild-type enzyme in the absence of MgATP and HCO(3)(-), however, the (15)N isotope effect is reduced 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 glutamine hydrolysis that is consistent with the isotope effects and with the catalytic properties of the enzyme is proposed.