title: Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4).
abstract: Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K(d)) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K(d) values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k(cat) values than that of WT (13.4 s⁻¹). The K(d) values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k(cat) values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k(cat) values of these interface mutants decreased as the K(d) values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.
authors: Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan.
authors: Yi-Liang
authors: YL
authors: Liu
authors: Yu-Hsiu
authors: YH
authors: Chiang
authors: Guang-Yaw
authors: GY
authors: Liu
authors: Hui-Chih
authors: HC
authors: Hung
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases