STEADY-STATE KINETICS OF GLUTAMINE CYCLOTRANSFERASE

The reaction mechanism of papaya latex glutamine cyclotransferase was studied using pH and temperature dependencies, a proton inventory tech nique, and molecular modeling. The pH-dependence of the Michaelis-Ment en 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 ch anged very slightly in the pH range between 4.5 and 10. The solvent ki netic isotope effect reflected a change in V-m while the proton invent ory was found to be linear with the fractionation factor of the exchan geable proton in the transition state of 0.785. The results were not c onsistent with an acyl-enzyme mechanism, but rather favored a simple i ntramolecular cyclization of the glutamine residue to the pyroglutamic acid residue. The mechanism proposed consists of the following main s teps: (i) intramolecular nucleophilic attack on the gamma-C=O carbon b y the nitrogen of the alpha-amino group, (ii) transfer of a proton fro m the alpha-amino group to the nitrogen of the amide group, facilitate d by an acidic group of the enzyme, and (iii) expulsion of the ammonia -leaving group promoted by this or another acidic enzyme group. (C) 19 94 Academic Press, Inc.