CATALYIS OF A PROTEIN-FOLDING REACTION - MECHANISTIC IMPLICATIONS OF THE 2.0 ANGSTROM STRUCTURE OF THE SUBTILISIN-PRODOMAIN COMPLEX
CATALYIS OF A PROTEIN-FOLDING REACTION - MECHANISTIC IMPLICATIONS OF THE 2.0 ANGSTROM STRUCTURE OF THE SUBTILISIN-PRODOMAIN COMPLEX
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DOI：
10.1021/bi00032a026
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发表时间：
1995-08-15
期刊：
影响因子：
2.9
通讯作者：
GALLAGHER, T
中科院分区：
文献类型：
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作者：
BRYAN, P;WANG, L;GALLAGHER, T
枯草杆菌蛋白酶的生物合成依赖于77个氨基酸的N-末端前结构域，其被自催化加工以产生酶的成熟形式[Ikemura，H.，Takagi，H.，& Inouye，M.（1987）J.Biol.Chem.262，7859 - 7864]。为了更好地了解前结构域在枯草杆菌蛋白酶折叠中的作用，我们已经确定了前结构域和枯草杆菌蛋白酶Sbt-70（一种用于促进折叠的突变体）之间的加工复合物的结构。前结构域本身在很大程度上是非结构化的，但当与枯草杆菌蛋白酶复合时，折叠成具有四链反平行β-折叠和两个三转角α-螺旋的紧凑结构。在25 ℃时，配合物的K_a为2 × 10~（8）M~（-1）。前结构域结合在枯草杆菌蛋白酶的两个平行表面α-螺旋上，并为两个螺旋的N-末端提供帽。前结构域的C-末端链结合在枯草杆菌蛋白酶底物结合裂缝中。虽然Sbt-70能够独立折叠，但在25 ℃下，在30 mM Tris-HCl（pH 7.5）中，前结构域将该过程加速> 10（7）M（-1）。体外折叠的突变体枯草杆菌蛋白酶的X-射线结构进行了比较，无论有或没有前结构域，并显示在任何情况下，相同的折叠stare是achieved。Sbt-70和前结构域的折叠反应的模型描述为以下平衡：P + S-a P-f-S-I P-f-S-f，其中S-u和P分别是Sbt-70和前结构域，它们在反应开始时基本上是非结构化的，P-f-S-I是部分折叠的Sbt-70和折叠的前结构域的碰撞复合物，P-f-S-f是折叠的Sbt-70和前结构域的复合物。前结构域结合的模式表明它通过稳定枯草杆菌蛋白酶中的中心α β α亚结构来催化枯草杆菌蛋白酶折叠。与该亚结构结合的前结构域可以对应于碰撞复合物P-f-S-I，其形成在双分子反应中是限速的[Strausberg，S.，亚历山大，P.，Wang，L.，美国，施瓦茨，F.，和Bryan，P.（1993）Biochemistry 32，8112 - 8119]。
Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864]. In order to better understand the role of the prodomain in subtilisin folding, we have determined the structure of the processed complex between the prodomain and subtilisin Sbt-70, a mutant engineered for facilitated folding. The prodomain is largely unstructured by itself but folds into a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices when complexed with subtilisin. The K-a of the complex is 2 x 10(8) M(-1) at 25 degrees C. The prodomain binds on subtilisin's two parallel surface alpha-helices and supplies caps to the N-termini of the two helices. The C-terminal strand of the prodomain binds in the subtilisin substrate binding cleft. While Sbt-70 is capable of independent folding, the prodomain accelerates the process by a factor of > 10(7) M(-1) of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 degrees C. X-ray structures of the mutant subtilisin folded in vitro either with or without the prodomain are compared and show that the identical folded stare is acheived in either case. A model of the folding reaction of Sbt-70 and the prodomain is described as the following equilibria: P + S-a P-f-S-I P-f-S-f, where S-u and P are Sbt-70 and prodomain, respectively, which are largely unstructured at the start of the reaction, P-f-S-I is a collision complex of a partially folded Sbt-70 and folded prodomain, and P-f-S-f is the complex of folded Sbt-70 and prodomain. The mode of prodomain binding suggests that it catalyzes subtilisin folding by stabilizing the central alpha beta alpha substructure in subtilisin. The prodomain bound to this substructure may correspond to the collision complex, P-f-S-I, whose formation is rate limiting in the bimolecular reaction [Strausberg, S., Alexander, P., Wang, L., Schwarz, F., & Bryan, P. (1993) Biochemistry 32, 8112-8119].