engleski

Dipeptidyl peptidase iii inhibition tests by metal dications

It has long been known that the peptidase activity of human dipeptidyl peptidase III (hDPP III) is inhibited by excess zinc ions. The aim of my paper is to determine the effects of different concentrations of metal dication: Zn2+, Mn2+, Co2+ and Cu2+ on the activity of hDPP III and to identify the inhibitory binding site of the metal. The existence of an inhibitory binding site was hypothesized based on a decrease in hDPP III activity at higher zinc concentrations and the similarity of hDPP III active site with those of carboxypeptidase A and thermolysin in whose crystallographic structures binding of another metal ion was observed in the immediate vicinity of the catalytically active ion. Inhibition of hDPP III by metal dications (Zn2+, Mn2+, Co2+ and Cu2+) is being investigated using various experimental and computational methods. For the purposes of experimental research, we prepare holoenzymes by incubating hDPP III with different concentrations of Zn2+, Mn2+, Co2+ and Cu2+ ions. In the samples thus prepared, metal-protein stoichometry is determined by high-resolution mass spectrometry with inductively coupled plasma (HRICP-MS) and microcalorimetric methods (isothermal titration calorimetry, ITC). Using the stopped-flow method, we monitor the decrease in hDPP III enzymatic activity caused by excess metal dications. Finally, using molecular dynamics combined with free binding energy calculus and quantum mechanical - molecular mechanical calculations we identified the zinc binding site and its effect on hDPP III structure and dynamics. Also, following the path of zinc ion exchange, we concluded that, as in the case of anthrax lethal factor, it is the so- called associative mode of modification described by relations: . enzyme ̶metal A + metal B ↔enzyme ̶metal A ̶metal B i. enzyme ̶metal A ̶metal B ↔enzyme ̶metal B +metal A . The results of this study allowed us to propose a mechanism by which the inhibitory zinc ion reduces enzyme activity.

hDppIII ; matal Zn ; Mn ; Cu ; Co ; inhibition

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