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

肽基精氨酸脱亚胺酶(蛋白质L-精氨酸亚氨水解酶，EC 3.5.3.15)是一种钙依赖的蛋白质调节酶，催化蛋白质中精氨酸残基的脱亚胺反应。使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。通过对[1-^&lt；14&gt；C]碘乙酰胺的动力学研究，我们发现两个碘乙酰胺分子与两个半胱氨酸残基结合导致了酶活性的完全丧失。底物Bz-L_=-Arg-O_--ET和辅酶因子Ca^&lt；2&gt；的共存对氨基乙酰胺的修饰具有完全的保护作用，其中任何一种配体都有部分保护作用。这些结果表明半胱氨酸基残基参与了催化反应。[1-^&lt；14&gt；C]碘乙酰胺修饰的酶在Sephadex G-50上用溴化氰氧化，得到两个面积大致相等的主要放射性峰，每个峰用赖氨酸内肽酶消化和C18反相高效液相色谱分离得到纯肽FI-P9和FII-P8。多肽Fi-p9含有一个修饰的半胱氨酸1个残基，而肽FII-p8含有4个修饰的半胱氨酸残基。手工埃德曼降解显示序列如下：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-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.