Haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4): evidence that general acid catalysis promotes efficient inactivation.
Dysregulated protein arginine deiminase (PAD) activity, particularly PAD4, has been suggested to play a role in the onset and progression of numerous human diseases, including rheumatoid arthritis (RA). Given the potential role of PAD4 in RA, we set out to develop inhibitors/inactivators that could be used to modulate PAD activity and disease progression. This effort led to the discovery of two mechanism-based inactivators, denoted F- and Cl-amidine, that inactivate PAD4 by the covalent modification of an active-site cysteine that is critical for catalysis. To gain further insights into the mechanism of inactivation by these compounds, the effect of pH on the rates of inactivation was determined. These results, combined with the results of solvent isotope effect and proton inventory studies, strongly suggest that the inactivation of PAD4 by F- and Cl-amidine proceeds by a multistep mechanism that involves the protonation and stabilization of the tetrahedral intermediate formed upon nucleophilic attack by the active-site cysteine, that is, Cys645. Stabilization of this intermediate would help to drive the halide-displacement reaction, which results in the formation of a three-membered sulfonium ring that ultimately collapses to form the inactivated enzyme. This finding-that protonation of the tetrahedral intermediate is important for enzyme inactivation-also suggests that, during catalysis, protonation of the analogous intermediate is required for efficient substrate turnover.
Knuckley
Bryan
B
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA.
Causey
Corey P
CP
Pellechia
Perry J
PJ
Cook
Paul F
PF
Thompson
Paul R
PR
D000143:Acids / Q000737:chemistry; D000578:Amidines / Q000737:chemistry*; D001172:Arthritis, Rheumatoid / Q000201:enzymology; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
D000143:Acids; D000578:Amidines; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D003545:Cysteine; C023731:acetamidine