The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.
The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.
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β-Arg-182（大肠杆菌 F1-ATP 酶中必需的催化位点残基）的作用。
DOI：
10.1021/bi990663x
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Nadanaciva,S;Weber,J;Senior,AE
大肠杆菌F1-ATP酶中的β-Arg-182（牛线粒体F1中的β-Arg-189）是靠近催化位点结合核苷酸的残基（Abrahams等人（1994）Nature 370, 621−628）。在这里，我们通过表征两个突变体βR182Q和βR182K来研究该残基的作用。纯化的 F1 的氧化磷酸化和稳态 ATP 酶活性因这两种突变而严重受损。使用 β-Trp-331 的荧光猝灭测量催化位点核苷酸结合参数，该荧光猝灭发生在核苷酸与来自 βR182Q/βY331W 和 βR182K/βY331W 双突变体的纯化 F1 结合时。结果发现，(a) β-Arg-182 与 MgATP 的 γ-磷酸盐相互作用，特别是在催化位点 1 和 2，(b) β-Arg-182 与 MgADP 的 β-磷酸盐或催化位点中的镁-核苷酸复合物的镁没有功能性相互作用，并且 (c) β-Arg-182 直接参与稳定化 的催化过渡态。在这些特征中，β-Arg-182 的作用类似于催化位点中另一个带正电的残基，即 Walker A 基序的保守赖氨酸 β-Lys-155。 β-Arg-182 的进一步作用被提出，即参与多位点催化所需的催化位点 β-α 亚基界面的构象变化。
β-Arg-182 inEscherichiacoliF1-ATPase (β-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994)Nature 370, 621−628). Here we investigated the role of this residue by characterizing two mutants, βR182Q and βR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of β-Trp-331 that occurred upon nucleotide binding to purified F1from βR182Q/βY331W and βR182K/βY331W double mutants. It was found that (a) β-Arg-182 interacts with the γ-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) β-Arg-182 has no functional interaction with the β-phosphate of MgADP or with the magnesium of the magnesium−nucleotide complex in the catalytic sites, and (c) β-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of β-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, β-Lys-155. A further role of β-Arg-182 is suggested, namely involvement in conformational change at the catalytic site β−α subunit interface that is required for multisite catalysis.