Deciphering the Catalytic Mechanism of the Zn Enzyme Glutaminyl Cyclase and the Deduction of Transition-State Analog Inhibitors

Zn(II)-dependent glutaminyl cyclase (QC) converts N-terminal glutamine or glutamate residues of peptides and proteins. The reaction product is in both cases N-terminal pyroglutamic acid (pyroGlu). In animals the glutaminyl cyclization is involved in posttranslational modification and activation of peptide-based hormone and chemokine precursors. Recently it was established that the driving force in neurodegenerative processes is the pyroGlu modification at the N-terminus of Aβ peptides processed by QC. This unveils the inhibition of QC as a strategy in AD treatment. The enzyme-specific inhibition is required in order to avoid noxious side effects. The elucidation of the reaction mechanism might make possible the development of mechanism-based QC inhibitors (e.g. transition-state analog compounds). Mechanistic and structural investigations were accomplished using the mitochondrial isoform of QC from Drosophila melanogaster. Regarding enzyme kinetic and structural properties, this enzyme is highly similar to the secretory hQC. Zn(II)-isoDromeQC wild type and variants harboring mechanism-relevant amino acids substitutes were heterologously expressed in E. coli. With the purified Zn(II) enzymes kinetic investigations were carried out including substrate specificity and pH-dependence studies. The substitution of the catalytically active Zn(II) ion into Co(II) yields an UV/Vis and EPR-active enzyme with almost the same enzymatic activity compared to the Zn(II) enzyme. The mentioned spectroscopic methods were used to obtain information about reaction trajectory, the binding modes of substrates and products. In addition the structure-function relationship of the enzyme was investigated in terms of the active center residues E190, D228 and D293. In particular the functions of these active center amino acids could be elucidated by successful crystallization trials. The analysis of the results reveal that the active-center amino acids D288, D293, E190 (side chains) and D293 (main chains) as well the metal ion are required for sufficient substrate binding. The function of D293 remains hypothetical due to the absence of a direct substrate-amino acid interaction. New information about the binding mode of N-terminal glutamate substrates were obtained by crystallization and EPR measurements. In contrast to four-coordinate Co(II) during the conversion of glutamine substrates, the binding of N-terminal glutamate provokes a five-coordination sphere of the metal ion. Both γ-carbonyl oxygens are non-covalently bound to the metal ion. Assuming that this conformation is catalytically inactive due to lack of a leaving hydroxyl group, it was proposed that the glutamyl cyclization takes up a proton from the solvent. Statements concerning this mechanism remain hypothetical for now