User:Alexander Rudecki/Sandbox 1: Difference between revisions

Within each subunit is 1 cysteine bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site, and are conserved in the human orthologue suggesting a pivotal role in catalysis<ref name="schilling"/>. However, when cysteines were replaced with alanines via site-directed mutagenesis, no kinetic differences were observed<ref name="main"/>. In contrast, this mutation did affect structural differences as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, and lack of its effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.

[[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 3. A comparison between the active sites of Drosophila melanogaster DromeQC crystalized either with a PBD150 inhibitor (right, 4F90) or without (left, 4FWU). Protein loops surrounding the active site are denoted in blue, and a key catalytic Zn²⁺ is shown as a grey sphere, chelated by three residues shown in light blue. The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

The active site of DromeQC is located on four loops that lack secondary structure (Figure 3). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn²⁺ exhibits trivalency (Figure 3). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn²⁺ was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. It is plausible that the amide oxygen of glutamine of peptide substrates chelate the zinc ion in a similar fashion, leading to position, polarization, and stabilization for cyclization.