Resolution of Reactive Center Structure of Peptidylarginine Deiminase, a Novel Protein Modulating Enzyme

肽基精氨酸脱亚胺酶（protein L-arginine de亚胺水解酶，EC 3.5.3.15）是一种Ca^<2+>依赖性蛋白调节酶，催化蛋白质中精氨酸残基的脱亚胺。利用碘乙酰胺和半胱氨酸特异性试剂对小鼠骨骼肌酶进行了化学修饰研究。从[1-^<14>C]碘乙酰胺的动力学研究中，我们发现两个碘乙酰胺分子与酶的两个半胱氨酸残基结合导致酶活性完全丧失。底物Bz-L__=-Arg-O__—Et和Ca^<2+>共存，酶的辅因子对吲哚乙酰胺修饰提供完全保护，上述配体均提供部分保护。这些结果表明半胱氨酸残基参与了催化作用。用溴化氰氧化的[1-^<14>C]碘乙酰胺修饰酶在Sephadex G-50上进行分选，发现两个面积近似相等的主放射性峰。用赖氨酸多肽酶酶切和C18反相HPLC层析得到纯化肽、FI-P9和fi - p8。肽Fi-p9含有1个半胱氨酸1修饰残基，肽fii-p8含有4个半胱氨酸基修饰残基。人工edman降解得到的序列为：Fi-P9， Ala-Ser- trp -Thr- trp -Gly-Pro- asn -Gly- cmcys -(Asx3,Glx2,Arg,Ala,Pro,Val,Ile,Leu3,Phe)lys和FII-P8， Thr-Pro- asn -Ile- leu -Pro-Val-Ser-Val- cal -(Asx2,Glx,CmCys4,Ser,Gly,Thr,Ala,Pro2,Val,Ile,Phe2)- met。

Peptidylarginine deiminase (protein L-arginine iminohydrolase, EC 3.5.3.15), A Ca^<2+>-dependent protein-modulating enzyme, catalyzes the deimination of arginyl residues in proteins. Chemical modification studies using iodoacetamide, and cysteine-specific reagent, have been carried out on the enzyme from mouse skeletal muscle. From kenetics studies with [1-^<14>C]iodoacetamide, we find that incorportion of two iodoacetamide molecule to two cysteinly residues of the enzyme resulted in complete loss of the enzymic activity. The co-existence of the substrate Bz-L__=-Arg-O__--Et and Ca^<2+>, cofactors for the enzyme offered complete protection against inodoacetamide modification, and either of the above ligands gave partial protection. These results indicate the involvement of the cysteinyl residues in catalysis. Fractionation of [1-^<14>C] iodoacetamide-modified enzyme oxidized with cyanogen bromide on Sephadex G-50, revealed two major radioactive peaks of approximately equal area.Digestion of each peak with lysylendopeptidase and chromatography on C18 reverse-phase HPLC resulted in pure peptides, FI-P9 and FII-P8. Peptide Fi-p9 contained one modified cysteiny 1 residue, while peptide fii-p8 contained four modified cysteinyl residues. manual edman degradation revealed the sequences as follows: Fi-P9, Ala-Ser-Trp-Thr-Trp-Gly-Pro-Asn-Gly-Cmcys-(Asx3,Glx2,Arg,Ala,Pro,Val,Ile,Leu3,Phe)lys and FII-P8, Thr-Pro-Asn-Ile-Leu-Pro-Pro-Val-Ser-Val-Cal-(Asx2,Glx,CmCys4,Ser,Gly,Thr,Ala,Pro2,Val,Ile,Phe2)-Met.