Lee R S, Wilke-Mounts S, Senior A E
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Arch Biochem Biophys. 1992 Sep;297(2):334-9. doi: 10.1016/0003-9861(92)90681-l.
Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.
大肠杆菌菌株AN718在F1F0 - ATP合酶中含有αS373F突变，该突变阻断了ATP合成（氧化磷酸化）和稳态F1 - ATP酶活性。含有αC373突变的回复菌株AN718SS2被分离出来，并显示在含有有限葡萄糖、琥珀酸盐或LB的液体培养基中，其生长产量表型高于野生型。发现从菌株AN718SS2纯化的F1具有野生型稳态ATP酶活性的30%和野生型氧化磷酸化活性的60%。ATP酶活性的叠氮化物敏感性和ADP诱导的结合金褐霉素荧光增强，这两者在αS373F突变体F1中均丧失，在αC373 F1中得以恢复。N - 乙基马来酰亚胺（NEM）能有效使αC373 F1稳态ATP酶失活，但对单点ATP酶无影响。αC373 F1稳态ATP酶的完全失活对应于每个F1（摩尔/摩尔）仅在三个α亚基之一中掺入一个NEM。NEM失活的酶显示出对叠氮化物不敏感的残余ATP酶活性以及ADP诱导的结合金褐霉素荧光增强的丧失。这些数据证实了这样一种观点，即在α373残基处放置一个庞大的氨基酸侧链（苯丙氨酰基或NEM衍生的半胱氨酰基）会阻断F1中的正催化协同作用。当三个α亚基中只有一个发生反应时NEM抑制稳态ATP酶这一事实表明存在一种循环催化机制。
