Aggeler R, Capaldi R A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Biol Chem. 1992 Oct 25;267(30):21355-9.
The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively. ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type. ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM). The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex. Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity. The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers. In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit. This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi. Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product. In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.
通过定点诱变改变了大肠杆菌F1 ATP酶（ECF1）的γ亚基，分别产生了五个不同的突变体，即γ-S8C、γ-S81C、γ-T106C、γ-S179C和γ-V286C。从其中四个突变体中分离得到的ECF1具有与用作对照的野生型同基因菌株相似的ATP酶活性，唯一的例外是从突变体γ-S81C中分离得到的酶，其ATP酶活性约为野生型的70%-80%。从各种突变体中分离得到的ECF1与N-(4-(7-(二乙氨基)-4-甲基香豆素-3-基))马来酰亚胺（CM）反应。荧光试剂掺入位于第8、106、179和286位的半胱氨酸残基中，但不掺入第81位，这表明这些半胱氨酸残基中哪些位于酶复合物中γ亚基的表面。用CM修饰第106位的半胱氨酸可激活该酶，修饰第8位的半胱氨酸会少量抑制ATP酶活性；然而，修饰第179或286位的半胱氨酸对酶活性没有影响。将四个带有反应性半胱氨酸的突变体与四氟苯基叠氮马来酰亚胺（TFPAMs）反应，TFPAMs是新型的光活化交联剂。在突变体γ-S8C中，γ亚基上引入的半胱氨酸与β亚基上的位点之间形成了交联。γ和β之间的这种交联取决于进行光解的核苷酸条件，与ATP + Mg2+或ATP + Mg2+ Pi相比，在ATP + EDTA条件下获得的交联产物迁移不同。β和γ之间的交联按交联产物的产量比例抑制ATP酶活性。在突变体γ-V286C中，γ上引入的半胱氨酸与α亚基之间形成了交联，在所有核苷酸条件下都是相同的，并且导致ATP酶活性受到抑制。
