Schmidt G, Senior A E
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Biochemistry. 1995 Aug 1;34(30):9694-9. doi: 10.1021/bi00030a006.
We introduced mutations at the highly-conserved residue Ser-339 in subunit beta of Escherichia coli F1-ATPase. The mutations beta S339Y and beta S339F abolished ATPase activity and impaired enzyme assembly. In contrast beta S339C F1 retained function to a substantial degree. N-Ethylmaleimide (NEM) at 0.2-0.3 mM inactivated beta S339C F1-ATPase by 80-95% in the presence of MgATP or MgADP but did not inactivate appreciably in absence of nucleotide or presence of EDTA. In absence of nucleotide, 0.7 mol of [14C-NEM] was incorporated into beta-subunits of 1.0 mol F1: in presence of MgATP the amount was 1.7 mol/mol, i.e. the introduced Cys residue became more accessible to reaction in the presence of MgATP. In the X-ray structure of F1 (Abrahams et al. (1994) Nature 370, 621-628) one of the catalytic nucleotide-binding domains is empty (on the "beta E subunit") and contains a cleft. Residue beta-339 lies within this cleft; the cleft does not occur in the other two beta-subunits. Our data are consistent with the conclusion that in wild-type enzyme under physiological conditions, MgATP or MgADP induce an enzyme conformation in which residue beta-Ser-339 becomes more exposed, possibly similar to the situation seen in the "beta E-subunit" in the X-ray structure.
我们在大肠杆菌F1 - ATP酶β亚基高度保守的丝氨酸-339位点引入了突变。βS339Y和βS339F突变消除了ATP酶活性并损害了酶的组装。相比之下，βS339C F1在很大程度上保留了功能。在存在MgATP或MgADP的情况下，0.2 - 0.3 mM的N - 乙基马来酰亚胺（NEM）使βS339C F1 - ATP酶失活80 - 95%，但在不存在核苷酸或存在EDTA时，未明显使其失活。在不存在核苷酸的情况下，每1.0摩尔F1的β亚基中掺入了0.7摩尔的[14C - NEM]；在存在MgATP的情况下，掺入量为1.7摩尔/摩尔，即引入的半胱氨酸残基在MgATP存在时更容易发生反应。在F1的X射线结构中（Abrahams等人（1994年）《自然》370, 621 - 628），一个催化性核苷酸结合结构域是空的（在“βE亚基”上），并且包含一个裂隙。β-339残基位于这个裂隙内；另外两个β亚基中不存在这个裂隙。我们的数据与以下结论一致：在生理条件下的野生型酶中，MgATP或MgADP诱导一种酶构象，其中β-丝氨酸-339残基变得更加暴露，可能类似于X射线结构中“βE亚基”的情况。
