Fluorescence stopped-flow study of the mechanism of nucleotide binding to myosin subfragment I.
Fluorescence stopped-flow study of the mechanism of nucleotide binding to myosin subfragment I.
复制标题
荧光停流研究核苷酸与肌球蛋白亚片段 I 结合的机制。
DOI：
10.1021/bi00591a003
复制
发表时间：
1979
期刊：
影响因子：
2.9
通讯作者：
H. Cheung
中科院分区：
文献类型：
--
作者：
F. Garland;H. Cheung
通过测定蛋白质与天然核苷酸(ATP或ADP)或它们的荧光类似物1.α-亚乙基腺苷5‘-三磷酸或二磷酸(eATP或EADP)反应时本征(色氨酸)荧光的增强或猝灭速率，研究了在有镁离子存在的情况下的(Si)。通过与完整SI[Sl(Mg)]和轻链缺失SI[Sl(EDTA)]的对比实验，研究了蛋白质的DTNB轻链(Lc2)对结合过程的影响。通过测量表观一级速率常数作为过量核苷酸浓度的函数得到的动力学等温线，对于天然核苷酸和类似物底物都是非线性的。三磷酸腺苷和腺苷二磷酸与完整SI反应的最大速率分别为39和32，而与SI(EDTA)(4 C，0.1Mkc1，5 mM氯化镁和0.1 mM二硫苏糖醇，pH 8.0)反应的最大速率分别为32和28 S。ATP和ADP等温线不符合Bagshaw等人的两步结合模型预测的矩形双曲线浓度依赖关系[Bagshaw，C.R“Eccleston，J.F.，Eackstein，F.，Goody，RS，Gutfreund，H.，&Trentham，Dr(1974)Biochem。虽然不能达到很高的浓度平台，但数据可以用双曲线拟合，SI(镁)和SI(EDTA)的最大速率分别为260和140 S~‘。EADP与完整的SI的反应呈明显的线性等温线，但在0.3 mM以上的核苷酸有两相示踪。相应的缺少轻链的实验产生了可分解为两个组分的两相痕迹：在15 0-2 0 0-S“1区趋于平稳的快组分和在高浓度时达到最大速率2 8 S”1的慢组分.综合这些实验结果，表明至少需要三个步骤来描述核苷酸脱除.在数据和计算机模拟计算的基础上，提出了一个表示Bagshaw-Trentham模型的简单扩展的机理.
(SI) in the presence of Mg2+ has been inves-tigated by measuring the rate of enhancement or of quenching of intrinsic (tryptophan) fluorescence when protein is reacted with eithernative nucleotides (ATP or ADP) or their fluor-escent analogues, 1. A^-ethenoadenosine 5'-tri-or-diphosphate (eATP or eADP). The influence of the DTNB light chain (LC2) of the protein on the binding process was studied by performing comparative experiments with intact SI [Sl (Mg)] and with light-chain deficient SI [Sl (EDTA)]. The kinetic isotherms, obtained by measuring the apparent first-order rate constant as a function of excess nucleotide concentration, were nonlinear for both native nucleotides and for analogue sub-strate. The maximum observed rate was 39 and 32 s" 1*, re-spectively, for ATP and ADP reacted with intact SI but 32 and 28 s" 1 with SI (EDTA)(4 C, 0.1 Mkc1, 5 mM MgCl2, and 0.1 mM dithiothreitol, pH 8.0). The ATP and ADP isotherms did not fit the rectangular hyperbolic concentration dependence predicted by the two-step binding model of Bagshaw et al.[Bagshaw, C. R „Eccleston, J. F., Eckstein, F., Goody, RS, Gutfreund, H., & Trentham, DR (1974) Biochem. J. 141, 351], Although high concentrationplateaus were not attained in the case of< ATP, the data could be fitted to hyperbolas, yielding maximum rates of 260 and 140 s~’with SI (Mg) and SI (EDTA), respectively. The reaction of eADP with intact SI gave an apparently linear isotherm; however, biphasic traces were obtained above 0.3 mM nucleotide. The corresponding light chain deficient experiments yielded bi-phasic traces which were resolvable into two components: a fast component which appears to level off in the 150-200-s” 1 region, and a slow component which attains a maximum rate of 28 s" 1 at high concentrations. The experimental results, taken together, indicate that a minimum of three steps is needed to describe nucleotidebinding. On the basis of the data and computersimulation calculations, a mechanism repre-senting a simple extension of the Bagshaw-Trentham model is proposed.