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

肽基精氨酸脱亚氨酶（蛋白L-精氨酸亚氨水解酶，EC 3.5.3.15），一种Ca^2+依赖性蛋白调节酶，催化蛋白质中乙酰基残基的脱亚氨作用。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-^ C]碘乙酰胺的动力学研究<14>中发现，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+）作为酶的辅因子，二者的共存对酶的亚氨基乙酰胺修饰提供了完全的保护作用，而上述两种配体中的任何一种都提供了部分保护作用。这些结果表明半胱氨酰残基参与催化。在<14>Sephadex G-50上用溴化氰氧化[1-^ C]碘乙酰胺修饰的酶，发现两个面积大致相等的主要放射性峰，用赖氨酰内肽酶消化每个峰，在C18反相HPLC上层析，得到纯肽FI-P9和FII-P8。肽Fi-p9含有一个修饰的半胱氨酸1残基，而肽fii-p8含有四个修饰的半胱氨酸残基。人工Edman降解揭示了如下序列：Fi-P9，Ala-Ser-Trp-Thr-Trp-Gly-Pro-Asn-Gly-Cmcys-（Asx 3，Glx 2，Arg，Ala，Pro，瓦尔，Ile，Leu 3，Phe）lys和FII-P8，Thr-Pro-Asn-Ile-Leu-Pro-Pro-瓦尔-Ser-瓦尔-Cal-（Asx 2，Glx，CmCys 4，Ser，Gly，Thr，Ala，Pro 2，瓦尔，Ile，Phe 2）-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.