Abstract
This study investigated the functional roles of the N-terminal Ca2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca2+-binding site of PAD4 were mutated to disrupt the binding of Ca2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k cat/K m,BAEE values were 0.02, 0.63 and 0.01 s−1mM−1 (20.8 s−1mM−1 for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k cat value of 0.3 s−1 (13.3 s−1 for wild-type), whereas D176A retained some catalytic power with a k cat of 9.7 s−1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k cat/K m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca2+ indicated that the conformational stability of the enzyme is highly dependent on Ca2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca2+ ions in the N-terminal Ca2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca2+ ions play critical roles in the full activation of the PAD4 enzyme.
Highlights
Peptidylarginine deiminase (PAD; protein-arginine deiminase, EC 3.5.3.15) is a Ca2+-dependent enzyme that catalyzes the conversion of arginyl to citrullyl residues in proteins, which is accompanied by the production of ammonia
For most peptidylarginine deiminase 4 (PAD4) alanine mutants, the Km,benzoyl-Larginine ethyl ester (BAEE) values were significantly elevated with decreased kcat and kcat/Km,BAEE values, which indicates that the catalytic efficiency of the enzyme was significantly reduced by these mutations
The unfolding curves of the integrated emission fluorescence for these two mutants were monophasic (Figure 5, right panels) with [Urea]0.5 values of 2.2 M and 2.4 M, respectively, which is similar to WT (2.6 M for WT; Table 3). These results demonstrate that most PAD4 mutants with low enzyme activity (N153A, N153Q, D155A, D155E and D157A) were less stable and more sensitive to urea than WT, which was indicated by the integrated emission fluorescence descending at the beginning of unfolding (Figure 4 and 5, right panels), and their intermediate states appeared unstable (Figure 4 and 5, left panels)
Summary
Peptidylarginine deiminase (PAD; protein-arginine deiminase, EC 3.5.3.15) is a Ca2+-dependent enzyme that catalyzes the conversion of arginyl to citrullyl residues in proteins, which is accompanied by the production of ammonia. The deiminase family is involved in a post-translational process called citrullination, which has significant effects on the physiological and functional properties of target proteins and plays a regulatory role in cell differentiation and development ([1,2,3], and references there in). PAD plays essential roles in cell differentiation [4], nerve growth [5,6], embryonic development [7], cell apoptosis [8,9,10] and gene regulation [11,12]. Various types of cancers are associated with PAD enzymes and their citrullinated targets [13,14,15,16,17]. Five isoforms of PAD (PAD1-4 and PAD6) have been characterized, and they have different tissue distributions [3,4,18–
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