Schultz Anders, Baltscheffsky Margareta
Arrhenius Laboratories, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius vag 10-12, S-10691 Stockholm, Sweden.
Biochim Biophys Acta. 2003 Dec 8;1607(2-3):141-51. doi: 10.1016/j.bbabio.2003.09.003.
The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.
来自光合细菌红螺菌（Rhodospirillum rubrum）的膜结合质子泵无机焦磷酸合酶/焦磷酸酶（H(+)-PPi合酶/H(+)-PPase）在大肠杆菌C43(DE3)细胞中实现了功能表达。基于该酶的新拓扑模型，选择预测位于膜附近或膜内的带电荷残基进行定点诱变。其中几个突变导致该酶几乎完全失活。四个突变残基似乎表现出质子转运的选择性损伤，因此可能参与焦磷酸水解与质子泵电耦合过程。其中两个突变，R176K和E584D，使酶对盐的耐受性增强。此外，前一个突变导致水解反应的米氏常数（K(m)）增加了一个数量级。本文讨论了这些结果及其对酶功能可能产生的影响。
