Abstract
Maximal rates of ATP hydrolysis catalyzed by F1-ATPase enzymes are known to involve strong positive catalytic site cooperativity. There are three potential catalytic nucleotide-binding sites on F1. Two important and unanswered questions are (i) whether all three potential catalytic sites must interact cooperatively to yield maximal rates of ATP hydrolysis and (ii) whether a cyclical three-site mechanism operates as suggested by several authors. We have studied these two questions here by measuring the ATPase activities of hybrid enzymes containing normal beta-, gamma-, delta-, and epsilon-subunits together with different combinations of mutant and normal alpha-subunits. The mutant alpha-subunits were derived from uncA401, uncA447, and uncA453 mutant E. coli F1-ATPase, in which positive cooperativity between catalytic sites is strongly attenuated by defined mis-sense mutations. Our data show that three normal catalytic sites are required to interact in order to achieve maximal ATPase rates and suggest that a cyclical mechanism does operate. Hybrid enzyme containing one-third mutant alpha-subunit and two-thirds normal alpha-subunits had substantial but submaximal activity, showing that cooperativity between three sites in a noncyclical fashion, or between pairs of sites, can achieve effective catalysis.
Highlights
The Properties of Hybrid F1-ATPaseEnzymes Suggest That a Cyclical Catalytic Mechanism Involving Three Catalytic Sites Occurs”
We have shown that positive cooperativity derived fromuncA401, uncA447a, nduncA453 mutant between catalytic sites is attenuated in three a-subunit(uncA)
E. coli Fl-ATPase,in which positive cooperativity be- missense mutants of E. coli such that, while ATP hydrolysis tween catalytic sites is strongly attenuated by definedrates under “unisite” conditions are normal, promotion of tmacmcalaciaiihtcsnlaia-ieaslnlyve-gtmsneiuoscemebncsuehami-ntxateiuinhtmstisisarhaamdtlarimodeAdnruesoTstuq.eOaPsbunausiotrsrtpeaedradenaa-trtsottaiuaiaetnbsletsb.uaehHurnnotayidwtcbstusrautibhdngmadegtnaeisxzttntwyihmmrtoohea-reeatldhctaeonicarrnottcdi-oryvsm-- naloscem(rwuta-utbahaLtui.lac,ynnhsi1ttisa9ss)r,8aetw4o,;hrSeticchesohpenneiiufoscosrtiruinmevadtelaulat“ycilmo,.e,tndhu1abl9elty8iusi4nimn)tut.eAecI”rnl4ear0ocea1ttatiie,cdosuhenndboocbfiAneetsdt4hwi4nen7eogte,thanarontecdaaec-uucmramatun(aAWtdlay4nit5@sit3ce-s ity, showing that cooperativity between threseites in site, is blocked (Wise et al, 1981;Senior et al, 1984;Perlin et a noncyclical fashion, or between pairs of sites, can al., 1984).The three mutantshave been sequencedand shown achieve effective catalysis
Summary
The Properties of Hybrid F1-ATPaseEnzymes Suggest That a Cyclical Catalytic Mechanism Involving Three Catalytic Sites Occurs”. Depolymerization of FI and repolymerization in the presence of added normal a-subunit were as described under“Materialsand Methods.”The total ATPase activity where y is the measured activity in pmol of Pi formed/ min/mg of F, proteininthe repolymerized mixture.
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