| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 94 === Peptidylarginine deiminase 4 (PAD4) is a Ca2+-dependent enzyme. The enzyme is highly correlated with rheumatoid arthritis. PAD4 gene has the single nucleotide polymorphisms (SNPs), four are exonic and three of them involved in amino acid substitutions: S55G, A82V, and A112G. We have established a series of SNP enzymes to identify the effects of SNPs on the PAD enzymes. The kcat value of A112G enzyme was higher than that of the wild-type enzyme by 2-fold, indicating that replacement of this residue elevated the catalytic capability of the enzyme. Notably, the kcat values of S55G/A112G and A82V/A112G and S55G/A82V/A112G were larger than that of wild-type enzyme, indicating that the A112G replacement causes the PAD enzyme more active. The activities of PAD4 were significantly stimulated by Ca2+ in a hyperbolic manner. Interestingly, the IC50 titration curves of Ca2+ for S55G, A112G, S55G/A112G, A82V/A112G, as well as S55G/A82V/A112G, displayed a biphasic curve indicating these SNP enzymes had two different binding affinities toward Ca2+. We also evaluated the Mg2+ inhibition to the PAD4 activity, and the results indicated the activities of these A112G-substituting enzymes were slighter affected by Mg2+ than the others. Furthermore, the enzyme conformations were analyzed with fluorescence quenching studies. Collectively, these findings suggest that the abnormal stimulation by Ca2+ for the enzyme activities of these A112G-substituting enzymes may be related to the
pathogenesis of rheumatoid arthritis.
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