第三, 四, 五號鈣離子結合位在人類第四型胜肽精胺酸去亞胺酶中所扮演的角色

Abstract

Peptidylarginine deiminase (PAD) is a post-translational modifying enzyme which catalyzes the conversion of protein arginine to citrulline in the presence of calcium. The PAD family has five isoforms and one of them, PAD4, is considered as the important factor of rheumatoid arthritis. The structures of PAD4 without or with calcium ions have been resolved by X-ray crystallography. There are five calcium ions occupying on the enzyme that are designated Ca1, Ca2, Ca3, Ca4, and Ca5, respectively. Calcium-binding induces PAD4 conformational change to form the active-site cleft. The Ca1 and Ca2, which are near to the catalytic site, are defined as critical calcium ions that assist in recognition of the substrate. The other three calcium ions are in the N-terminal domain away from the catalytic site. The functional roles of Ca3, Ca4, and Ca5 are still undefined. Here we use site-directed mutagenesis and enzyme kinetic analysis to interpret the role of Ca3, Ca4, and Ca5 binding sites. The ten residues, N153, D155, D157, D165, D168, E170, D176, D179, E252, and D388, are replaced by alanine or amide amino acid to interrupt the calcium binding on this region. These mutants at the Ca3 and Ca4 binding sites show higher Km and lower kcat than WT, suggesting that Ca3 and Ca4 are essential for PAD4 catalysis. Our fluorescence studies further demonstrate that these mutants display different conformations as compared with WT. The mutants of D168, E170, and E252, which participate the Ca5 binding, have weaker effects on Km and lower kcat than Ca3 and Ca4. Our findings show that Ca3 and Ca4, although far away from the active site, still have significant influence on substrate binding and calcium cooperativity.