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

新型蛋白质调节酶肽基精氨酸脱亚氨酶反应中心结构的解析

• 批准号: 62560071
• 负责人: TAKAHARA Hidenari
• 金额: $ 1.22万
• 依托单位: Ibaraki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62560071/
• 关键词: Peptidylarginine deiminase; Structure of reactive center; Chemical Modification of reactive center; ヨードアセトアミドによる活性中心残基の修飾; ヨードアセトアミドによる活性中心システイン残基の修飾

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.

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。

项目成果

高原英成: 日本農芸化学会誌. 62. 1125-1126 (1988)

Takahara, E.：日本农业化学学会杂志 62. 1125-1126 (1988)

Hidenari Takahara; Kiyoshi Sugawara: "Studies on Peptidylarginine Deiminase: Resolution of Reactive Center by Chemical Modification" Nippon Nougeikagaku Kaishi. 62. 648 (1988)

高原英成；

高原英成、菅原潔: 日本農芸化学会誌. 62. 648 (1988)

高原秀成、菅原清：日本农业化学学会杂志 62. 648 (1988)。

Hidenari Takahara: "Resolution of Reactive Center Structure of Peptidylarginine Deiminase, a Novel Protein Modulating Enzyme" Nippon Nougeikagaku Kaishi. 62. 1125-1126 (1988)

Hidenari Takahara：“肽基精氨酸脱亚胺酶（一种新型蛋白质调节酶）反应中心结构的解析”Nippon Nougeikagaku Kaishi。

Hidenari Takahara: Journal of Biachemistry.

高原秀成：生物化学杂志。