Hyndman D J, Milgrom Y M, Bramhall E A, Cross R L
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center, Syracuse 13210.
J Biol Chem. 1994 Nov 18;269(46):28871-7.
Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi. No binding of GDP at non-catalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity. However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone. For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used. With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase. Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants.
核苷酸耗尽的EcF1在非催化位点最多可结合两个GTP、ATP或ADP，而所有三个位点只能由核苷二磷酸和三磷酸的组合填充。MgPPi可阻止GTP的结合并显著减缓ATP的结合，这表明非催化位点也能结合PPi。在非催化位点未检测到GDP的结合。GTP从非催化位点解离的速率较慢（t1/2 = 175分钟），EDTA、MgPPi、MgADP或EDTA/ATP可使其增加2至8倍，但ATP水解条件下可使其增加23倍。去除抑制性ε亚基和内源性核苷酸的EcF1进行ATP水解时，会出现活性爆发。然而，如果预先与MgADP孵育则会出现滞后现象，而单独与Mg2+预先孵育时则不会。对于含有内源性抑制性ADP的ε亚基缺失的EcF1，与ATP再生系统预先孵育会导致活性爆发，而对照组则出现滞后现象。同样的酶形式在ATP水解过程中随着Mg2+浓度的增加表现出显著抑制，但使用其他NTP底物时抑制水平较低。对于五亚基酶，叠氮化物量的增加会导致抑制水平升高，同时对快速追踪具有抗性的结合核苷酸量相应增加。如2-叠氮基-ADP的共价掺入所示，可被叠氮化物捕获的核苷酸结合在催化位点。结果表明，配体特异性可能不是区分催化位点和非催化位点的可靠方法，并且在EcF1突变体的动力学分析中应考虑MgADP的抑制作用。
