Park S, Ajtai K, Burghardt T P
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Biochemistry. 1997 Mar 18;36(11):3368-72. doi: 10.1021/bi9624999.
Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBeFx-S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPA1F4-S1 and MgADPScFx-S1) falling in between in the order MgADPBeFx > MgADPA1F4 > MgADPScFx > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the gamma-phosphate analog metal from the oxygen connecting it to the beta-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X-ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the beta- and the gamma-phosphate, stresses the active site of S1 through protein-nucleotide contacts at the gamma-phosphate and nucleotide base. The stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure.
兔骨骼肌肌球蛋白亚片段1（S1）中色氨酸510（Trp510）荧光的丙烯酰胺猝灭表明，在被困于S1活性位点的核苷酸或核苷酸类似物存在下，包含高反应性硫醇（SH1）和Trp510的探针结合裂隙的构象[Park等人（1996年），《生物化学与生物物理学报》1296，1 - 4]。Trp510猝灭效率表明，在被困有氟化铍的MgADP（MgADPBeFx - S1）存在下，探针结合裂隙略有闭合；在被困有钒酸盐的MgADP（MgADPVi - S1）存在下，闭合最紧密；被困有氟化铝和氟化钪的MgADP（MgADPA1F4 - S1和MgADPScFx - S1）则介于两者之间，顺序为MgADPBeFx > MgADPA1F4 > MgADPScFx > MgADPVi。这些核苷酸类似物与MgATP在水解过程中的顺序结构变化相对应，氟化铍出现在ATP酶循环的最早阶段。γ - 磷酸类似物金属与ADP中连接它与β - 磷酸的氧之间的分离距离与裂隙闭合程度的相关性表明，核苷酸过渡态中的这一距离决定了探针结合裂隙的构象。Trp510猝灭效率还作为被困有钒酸盐的Mg - 核苷酸二磷酸（MgNDPVi - S1）碱基部分的函数进行了测量。在天然底物NDP存在下，裂隙闭合程度最大，顺序为MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi，MgADPVi和MgCDPVi之间差异很小。这些数据符合相应三磷酸核苷酸支持肌肉纤维中力产生的有效性顺序[Pate等人（1993年），《生物化学杂志》268，10046 - 10053]。在纤维和S1中，似乎ADP和CDP碱基的6位氨基是将核苷酸正确锚定在活性位点所必需的，如X射线晶体学研究[Fisher等人（1995年），《生物化学》34，8960 - 8972]所暗示的，可能是在酪氨酸135处。最后，Trp510猝灭效率作为被困于S1活性位点与ADPV i结合的二价阳离子大小的函数进行了测量。这些数据未显示出相关性，表明二价阳离子不参与从活性位点到探针结合裂隙的影响传播。上述实验表明，水解过程中ATP构象的变化，以β - 和γ - 磷酸之间距离的增加为参数，通过γ - 磷酸和核苷酸碱基处的蛋白质 - 核苷酸接触对S1的活性位点施加压力。横桥中由压力诱导的应变可能是ATP中的能量转移到肌球蛋白结构的机制。
