Investigating the geminal diamine intermediate of Yersinia pestis arginine decarboxylase with substrate, product, and inhibitors using single wavelength stopped-flow spectroscopy.
The reaction mechanism of Yersinia pestis arginine decarboxylase has been investigated using a series of substrate, product, and inhibitors. Using single wavelength stopped-flow spectroscopy, novel mechanistic features were noted in the presence of the product, agmatine. By focusing on the excitation and emission wavelengths of the geminal diamine intermediate, we were able to monitor the formation and decay of two different geminal diamine species. Experiments revealed that the enzyme exists in two different conformational states--one that binds ligand and one that does not. The on and off rates for the conversion between the two conformational states was determined to be 390 s-1 and 880 s-1, respectively. The KD for agmatine binding was 6 mM. In addition, experiments revealed a pH-dependent conversion between two states of the enzyme. The deprotonated form of the enzyme binds ligand more slowly than the protonated form. The rates for the formation of the geminal diamine and external aldimine in this pathway were determined to be 25 and 4 s-1, respectively. There is also a slow interconversion between the protonated and deprotonated enzymes that has a pKa of approximately 8.0. Finally, the formation of the geminal diamine was determined to be Mg2+-dependent.
Counts
Korie G
KG
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, USA.
Wong
Isaac
I
Oliveira
Marcos A
MA
D000376:Agmatine / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003959:Diamines / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D015010:Yersinia pestis / Q000201:enzymology* / Q000235:genetics
D003959:Diamines; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000376:Agmatine; D001120:Arginine; D002262:Carboxy-Lyases; C019722:arginine decarboxylase