Structural and functional characterization of a peptide processing enzyme: Human glutaminyl cyclase

Rachell Eschette Booth


Peptides specifically neurotransmitters and hormones are processed into their mature form along the secretory pathway by different processing enzymes. Glutaminyl cyclase (QC) plays an important role in this maturation by cyclizing N-terminal glutamine to pyroglutamyl residue to produce biologically active peptides. In an effort to determine the mechanism of action and important structural characteristics of QC, human QC has been expressed in Drosophilia Schneider 2 cells (DES hQC) using a metallothione promoter and a signal sequence directing secretion. In this system, active protein is collected from the media at levels approaching 50 ug/mL. This recombinant form of the protein has been partially purified using anion-exchange chromatography and characterized to validate its representation of the native form. Complete purification of DES hQC and partial purification of native bovine pituitary QC was achieved with the use of a Reactive Blue 4 agarose affinity column. The pH activity optimum and kinetic constants for DES QC were found to be similar to that of the native bovine pituitary enzyme. Thus, the recombinant QC will be a good representation for the native protein in further structural studies. Gel shift assays indicated that the DES QC was glycosylated and contains a disulfide-bonded cysteine bridge. Mutational analysis of the predicted glycosylation site resulted in an active QC form despite the absence of N-glycans. Structural characterization for the pure protein using circular dichroism showed that hQC contains α-helix and β-sheet structures. These results corroborate with the secondary structures seen in a PRODOM predicted model of hQC, which is based on its structural homology with known protein crystal structures. In this case, the highest structural homology relation was to Aeromonas proteolytica aminopeptidase, a protease that also recognizes the N-terminal of proteins. Potential hQC active site residues were also identified using this predicted model as well as the amino acid sequence alignment for QC proteins across mammalian species. Several of these residues within hQC including Glu 201, Glu 202, Asp 159, and Asp 248 were individually mutated and the resulting mutant proteins characterized. Kinetic analysis showed that mutations in Glu 201 and Glu 202 residues resulted in completely inactive protein; thus these residues are essential for activity. However, the activity of the Asp 159 mutant was only partially decreased and thus this residue may be involved in catalysis. The Asp 248 mutation had no effect on activity. These results indicate that glutamate residues play a role in catalysis, suggesting an acid/base catalytic mechanism, and that the predicted model was a good representation of the QC structure.