Murphy C T, Spudich J A
Departments of Biochemistry and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
Biochemistry. 1999 Mar 23;38(12):3785-92. doi: 10.1021/bi9826815.
We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.
我们感兴趣的是溶剂暴露的、对蛋白水解敏感的表面环在肌球蛋白功能中所起的作用。25 - 50K环，即环1，靠近ATP结合位点，而50 - 20K环（环2）位于肌动蛋白结合位点。通过嵌合研究，我们发现环1影响ADP的释放[墨菲，C.T.，和斯普迪奇，J.A.（1998年）《生物化学》37卷，6738 - 6744页]，而环2影响肌动蛋白激活的ATP酶活性[上田，T.Q.-P.等人（1994年）《自然》368卷，567 - 569页]。在本文所述的研究中，我们发现肌动蛋白激活的ATP酶活性的催化常数（kcat）因环2的替换而改变，其方式反映了供体肌球蛋白相对的肌动蛋白激活的ATP酶活性。此外，环2的变化在有核苷酸和无核苷酸的情况下均会影响肌球蛋白对肌动蛋白的亲和力。稳态前研究以及ATP酶和亲和力数据表明，虽然环2不影响肌球蛋白与核苷酸之间的相互作用，但它在决定肌球蛋白在各种核苷酸状态下对肌动蛋白的亲和力以及在允许磷酸释放的限速转变中发挥作用。
