Löbau S, Weber J, Senior A E
Department of Biochemistry and Biophysics, University of Rochester Medical Center, NY 14642, USA.
FEBS Lett. 1997 Mar 3;404(1):15-8. doi: 10.1016/s0014-5793(97)00077-x.
Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites were filled in the E. coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure. However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e.g. Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.
我们使用定点色氨酸荧光技术，在先前用于牛线粒体酶结晶和X射线结构分析的条件下 [亚伯拉罕斯等人（1994年）《自然》370, 621 - 628]，研究了大肠杆菌F1 - ATP酶催化位点的核苷酸占据情况。我们发现，在这些条件下（250微摩尔MgAMPPNP加5微摩尔MgADP），大肠杆菌酶的三个催化位点中只有两个被占据，这与牛F1 X射线结构中的报道一致。然而，随后添加生理浓度的MgATP很容易填满第三个催化位点。因此，X射线结构中看到的酶形式是由于它是在亚饱和核苷酸条件下获得的。数据表明，X射线结构与一种催化机制相符，即所有三个F1 - ATP酶催化位点都必须填满MgATP才能启动稳态水解 [例如，韦伯和西尼尔（1996年）《生物化学与生物物理学报》1275, 101 - 104]。数据进一步证明，定点色氨酸荧光技术可为F1晶体学研究提供有价值的支持。
