Turina P, Capaldi R A
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Biol Chem. 1994 May 6;269(18):13465-71.
Four mutants of the Escherichia coli F1ATPase, gamma S8-C, gamma T106-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue. With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored. The results obtained with the mutants gamma S8-C and gamma T106-C are similar. In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP. The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching. The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the gamma-subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites. No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites. The fluorescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon-free E. coli F1ATPase, although such enzyme preparations are highly active ATPases. We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling.
通过定点诱变在大肠杆菌F1ATP酶的γ亚基不同位点引入半胱氨酸的四个突变体，即γS8 - C、γT106 - C、γS179 - C和γV286 - C，在选择性标记引入的半胱氨酸残基的条件下，与荧光试剂N - (4 - 7 - (二乙氨基) - 4 - 甲基香豆素 - 3 - 基) - 马来酰亚胺(CM)反应。对于每个突变体，都监测了核苷酸结合对探针荧光的影响。用突变体γS8 - C和γT106 - C得到的结果相似。在这两种情况下，向催化位点无核苷酸的酶中添加AMP.PNP或ATP时，会出现光谱位移和荧光强度变化，而添加ADP时未观察到荧光光谱变化。用ATP获得的荧光光谱变化是瞬时的，包括最初的快速荧光增强，随后是荧光猝灭。在单位点催化条件下，这些ATP诱导的荧光变化动力学以及通过ATP结合速率和Pi形成速率监测的ATP水解动力学是相同的，这表明荧光探针测量的γ亚基构象变化是由催化位点的ATP水解驱动的。当CM结合在179位的半胱氨酸时，未观察到核苷酸依赖性荧光变化。当CM结合在286位时，观察到核苷酸依赖性荧光变化，但这些变化似乎反映了非催化位点结合ADP或ATP引起的结构变化。在无亚基ε的大肠杆菌F1ATP酶中未观察到突变体γS8 - C和γT106 - C中观察到的荧光变化，尽管这种酶制剂是高活性的ATP酶。我们得出结论，荧光探针监测的结构变化是构象偶联的一部分，通过这种偶联，催化位点事件与质子通道相连。
