PROTEOLYSIS OF THE CYTOCHROME-D COMPLEX WITH TRYPSIN AND CHYMOTRYPSIN LOCALIZES A QUINOL OXIDASE DOMAIN
PROTEOLYSIS OF THE CYTOCHROME-D COMPLEX WITH TRYPSIN AND CHYMOTRYPSIN LOCALIZES A QUINOL OXIDASE DOMAIN
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DOI：
10.1021/bi00228a007
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发表时间：
1991-04-09
期刊：
影响因子：
2.9
通讯作者：
GENNIS, RB
中科院分区：
文献类型：
--
作者：
DUEWEKE, TJ;GENNIS, RB
细胞色素d复合体是大肠杆菌需氧呼吸链中的两个亚单位，膜结合的末端氧化酶。该酶催化泛喹酚的两电子氧化和氧的四电子还原为水。以前的工作表明，泛喹酚氧化的位置被胰酶有限的蛋白分解选择性地灭活，该酶在亚基I内的一个位置裂解。这项工作扩展到表明在有限的胰凝乳蛋白酶蛋白分解复合体中也观察到类似的现象。无论是在非变性洗涤剂中使用纯化的氧化酶，还是在蛋白脂质体中重组，或者使用大肠杆菌的球体作为蛋白质降解的底物，切割模式都是相似的。因此，蛋白水解酶敏感位点在细胞中是周质的。用N-末端测序和免疫印迹的方法对蛋白降解产物进行了鉴定。数据表明，泛喹酚氧化酶活性的失活是由于特定残基的裂解与先前定义为Q环的亲水性区域的裂解所致。这一结构域已经被抑制的单抗用于泛喹酚的氧化。对酶裂解的酶进行的电化学和高效液相分析表明，该酶的四级或三级结构没有全局变化。Q环很可能直接参与在膜的周质表面附近形成部分泛喹酚结合部位。
The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane.