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National Repository of Grey Literature 1 records found  Search took 0.00 seconds. 
The effect of Ca2+ ion on the enzymatic activity of human glutamate carboxypeptidase II
 Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Hlouchová, Klára (referee)
Human glutamate carboxypeptidase II (GCPII, EC 3.4.17.24) is a homodimeric membrane glycoprotein. GCPII has been studied as a marker of prostate carcinoma and a therapeutic target of neurodegenerative disorders. The extracellular region of the protein is composed of three domains, apical, protease and C-terminal. There are two zinc ions in the active site that are essential for the enzymatic activity. A calcium ion is located between apical and protease domains near the dimeric interface approximately 20 Å away from the active site. Consequently, the Ca2+ ion in unlikely to participate in substrate hydrolysis. The aim of this thesis is to elucidate the function of Ca2+ in GCPII using a combination of molecular-biological, biochemical and biophysical approaches. To this end we prepared series of GCPII variants with mutations in calcium-coordinating amino acids. The mutant constructs were expressed in insect S2 cells and purified by combination of affinity and size exclusion chromatography. Enzymatic activity and thermostability of the mutants were decreased significantly. Furthermore, mutated proteins were aggregation prone and formed a monomeric GCPII species. Our results thus show that Ca2+ ion plays an essential role in proper GCPII folding as well as the formation of a homodimer molecule that is...
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