Steady-state kinetics of glutamine cyclotransferase.

The reaction mechanism of papaya latex glutamine cyclotransferase was studied using pH and temperature dependencies, a proton inventory technique, and molecular modeling. The pH-dependence of the Michaelis-Menten parameters showed that the published pH dependence of the enzyme "activity" was mainly the result of pH-dependent change of the active (unprotonated) substrate concentration. The enzyme activity as such changed very slightly in the pH range between 4.5 and 10. The solvent kinetic isotope effect reflected a change in Vm while the proton inventory was found to be linear with the fractionation factor of the exchangeable proton in the transition state of 0.785. The results were not consistent with an acyl-enzyme mechanism, but rather favored a simple intramolecular cyclization of the glutamine residue to the pyroglutamic acid residue. The mechanism proposed consists of the following main steps: (i) intramolecular nucleophilic attack on the gamma-C = O carbon by the nitrogen of the alpha-amino group, (ii) transfer of a proton from the alpha-amino group to the nitrogen of the amide group, facilitated by an acidic group of the enzyme, and (iii) expulsion of the ammonia-leaving group promoted by this or another acidic enzyme group.