Ahmad Zulfiqar, Senior Alan E
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
J Biol Chem. 2005 Jul 29;280(30):27981-9. doi: 10.1074/jbc.M503955200. Epub 2005 Jun 6.
This paper presents a study of the role of positive charge in the P(i) binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P(i) binding sites of proteins generally and in the P(i) binding site of the betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-246 retained P(i) binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.
本文介绍了一项关于正电荷在大肠杆菌ATP合酶的磷酸根（Pi）结合位点中作用的研究。ATP合酶负责通过氧化磷酸化进行ATP驱动的质子外排和ATP合成。一般来说，精氨酸残基在蛋白质的Pi结合位点中具有很高的出现概率，特别是在ATP合酶的βE催化位点的Pi结合位点中。去除天然的β精氨酸-246（βR246A突变体）会消除Pi结合；通过将β天冬酰胺-243或α苯丙氨酸-291中的任何一个残基突变为精氨酸，可恢复Pi结合。在X射线结构中，这两个残基都位于靠近β精氨酸-246的Pi结合位点中。在存在β精氨酸-246的情况下，在β-243或α-291处插入一个额外的精氨酸可保留Pi结合，但在两个位置同时插入两个额外的精氨酸则会消除Pi结合。使用氟铝酸盐和氟钪等磷酸盐类似物来测量过渡态稳定性。在βR246A中去除β精氨酸-246几乎导致过渡态稳定性完全丧失，但在βN243R/βR246A和αF291R/βR246A中实现了部分挽救。在存在β精氨酸-246的情况下，β精氨酸-243或α精氨酸-291的效果较差；αF291R/βN243R与天然β精氨酸-246的组合与βR246A一样有害。数据表明，静电相互作用是催化位点βE中初始Pi结合以及后来在过渡态复合物中的一个重要组成部分。然而，由于没有一个突变体在生长测试、ATP驱动的质子泵浦或ATP酶活性测定中表现出显著功能，显然催化位点精氨酸残基的特定立体化学相互作用至关重要。
