title: Kinetic characterization of protein arginine deiminase 4: a transcriptional corepressor implicated in the onset and progression of rheumatoid arthritis.
abstract: Protein arginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the posttranslational conversion of arginine to citrulline (Arg --> Cit) in a number of proteins, including histones. While the gene encoding this enzyme has been implicated in the pathophysiology of rheumatoid arthritis (RA), little is known about its mechanism of catalysis, its in vivo role, or its role in the pathophysiology of RA; however, recent reports suggest that this enzyme can act as a transcriptional corepressor for the estrogen receptor. Herein, we report our initial kinetic and mechanistic characterization of human PAD4. Specifically, these studies confirm that PAD4 catalyzes the hydrolytic deimination of Arg residues to produce Cit and ammonia. The metal dependence of PAD4 has also been evaluated, and the results indicate that PAD4 activity is highly specific for calcium. Calcium activation of PAD4 catalysis exhibits positive cooperativity with K(0.5) values in the mid to high micromolar range. Evidence indicating that calcium binding causes a conformational change is also presented. Additionally, the steady-state kinetic parameters for a number of histone H4-based peptide substrates and benzoylated Arg derivatives have been determined. K(m) values for these compounds are in the high micromolar to the low millimolar range with k(cat) values ranging from 2.8 to 6.6 s(-)(1). The ability of PAD4 to catalyze the deimination of methylated Arg residues has also been evaluated, and the results indicate that these compounds are poor PAD4 substrates (V/K <or= 31.3 M(-)(1) s(-)(1)) in comparison to other substrates. These findings suggest that the full-length enzyme does not catalyze this reaction in vitro and possibly in vivo either. Collectively, the studies described herein will provide a firm foundation for the future development of PAD4 selective inhibitors.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Kearney
authors.forename: Monica
authors.initials: M
authors.lastname: Bhatia
authors.forename: Nelroy G
authors.initials: NG
authors.lastname: Jones
authors.forename: Luo
authors.initials: L
authors.lastname: Yuan
authors.forename: Mary C
authors.initials: MC
authors.lastname: Glascock
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Catchings
authors.forename: Michiyuki
authors.initials: M
authors.lastname: Yamada
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
mesh_terms: D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000201:enzymology* / Q000503:physiopathology; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D018450:Disease Progression; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006657:Histones; D010455:Peptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D000641:Ammonia; D001120:Arginine; C009727:benzoylarginine ethyl ester; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D002118:Calcium