Cheng X, Shaltiel S, Taylor S S
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
Biochemistry. 1998 Oct 6;37(40):14005-13. doi: 10.1021/bi981057p.
Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKI resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.
当底物结合时，环磷酸腺苷依赖性蛋白激酶（cAPK）的催化亚基（C）会发生显著的诱导构象变化，从而引发催化作用。对于游离的脱辅基酶，平衡有利于更开放且可塑性更强的构象，而C、MgATP和一个20个残基的抑制肽[PKI(5 - 24)]形成的三元复合物则采用紧密且封闭的构象[郑，J.等人（1993年），《蛋白质科学》2，1559]。目前尚不清楚是单独一种配体的结合导致了这种构象转换，还是两种配体都需要。此外，催化亚基协同结合MgATP和抑制肽。这种协同作用的结构基础目前也尚未明确。利用铁 - 乙二胺四乙酸介导的蛋白质足迹技术，通过绘制C的溶剂可及表面和结构动力学，探究了与MgATP和热稳定蛋白激酶抑制剂（PKI）结合相关的构象变化。游离酶的构象与三元复合物明显不同。此外，单独MgATP的结合会引发广泛的局部和全局构象变化，包括富含甘氨酸的环、连接小和大亚基的连接区、催化环、Mg2 +定位环、激活环和F螺旋。这些变化与在三元复合物中观察到的变化相似，与从开放构象到更封闭构象的转变一致，可能反映了与催化和产物释放相关的运动。相比之下，C.PKI的足迹模式类似于游离的C，表明构象变化极小。MgATP的结合通过将平衡转移到更封闭的构象，“使”酶处于准备好对接PKI的状态，并为MgATP和PKI之间的协同作用提供了解释。
