title: Protein arginine deiminase 4: evidence for a reverse protonation mechanism.
abstract: The presumed role of an overactive protein arginine deiminase 4 (PAD4) in the pathophysiology of rheumatoid arthritis (RA) suggests that PAD4 inhibitors could be used to treat an underlying cause of RA, potentially offering a mechanism to stop further disease progression. Thus, the development of such inhibitors is of paramount importance. Toward the goal of developing such inhibitors, we initiated efforts to characterize the catalytic mechanism of PAD4 and thereby identify important mechanistic features that can be exploited for inhibitor development. Herein we report the results of mutagenesis studies as well as our efforts to characterize the initial steps of the PAD4 reaction, in particular, the protonation status of Cys645 and His471 prior to substrate binding. The results indicate that Cys645, the active site nucleophile, exists as the thiolate in the active form of the free enzyme. pH studies on PAD4 further suggest that this enzyme utilizes a reverse protonation mechanism.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Knuckley
authors.forename: Monica
authors.initials: M
authors.lastname: Bhatia
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites; D002956:Citrulline / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011522:Protons; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D002956:Citrulline; D006639:Histidine; D001120:Arginine; C009727:benzoylarginine ethyl ester; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D003545:Cysteine