May A, Vas M, Harlos K, Blake C
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Proteins. 1996 Mar;24(3):292-303. doi: 10.1002/(SICI)1097-0134(199603)24:3<292::AID-PROT2>3.0.CO;2-J.
The crystal structure of a ternary complex of pig muscle phosphoglycerate kinase (PGK) containing 3-phosphoglycerate (3-PG) and manganese adenylylimidodiphosphate (Mn AMP-PNP) has been determined and refined at 2.0 A resolution. The complex differs from the true substrate ternary complex only in the presence of an imido- rather than an oxylink between beta- and gamma-phosphates of the bound nucleotide. The 3-PG is bound in a similar manner to that observed in binary complexes. The nucleotide is bound in a similar manner to Mg ADP except that the metal ion is coordinated by all three alpha-, beta-, and gamma-phosphates, but not by the protein. The gamma-phosphate, which is transferred in the reaction, is not bound by the protein. One further characteristic of the ternary complex is that Arg-38 moves to a position where its guanidinium group makes a triple interaction with the N-terminal domain, the C-terminal domain, and the 1-carboxyl group of the bound 3-PG. Although a hinge-bending conformation change is seen in the ternary complex, it is no larger than that observed in the 3-PG binary complex. To reduce that distance between two bound substrates to a value consistent with the direct in-line transfer known to occur in PGK, we modeled the closure of a pronounced cleft in the protein structure situated between the bound substrates. This closure suggested a mechanism of catalysis that involves the "capture" of the gamma-phosphate by Arg-38 and the N-terminus of helix-14, which has a conserved Gly-Gly-Gly phosphate binding motif. We propose that nucleophilic attack by the 1-carboxyl group of the 3-PG on the gamma-phosphorus follows the capture of the gamma-phosphate, leading to a pentacoordinate transition state that may be stabilized by hydrogen bonds donated by the NH groups in the N-terminus of helix 14 and the guanidinium group of Arg-38. During the course of the reaction the metal ion is proposed to migrate to a position coordinating the alpha- and beta-phosphates and the carboxyl group of Asp-374. The mechanism is consistent with the structural information from binary and ternary substrate complexes and much solution data, and gives a major catalytic role to Arg-38, as indicated by site-directed mutagenesis.
已测定并在2.0埃分辨率下精修了含有3-磷酸甘油酸（3-PG）和锰腺苷亚氨基二磷酸（Mn AMP-PNP）的猪肌肉磷酸甘油酸激酶（PGK）三元复合物的晶体结构。该复合物与真正的底物三元复合物的区别仅在于结合核苷酸的β-和γ-磷酸之间存在亚氨基而非氧基连接。3-PG的结合方式与在二元复合物中观察到的相似。核苷酸的结合方式与Mg ADP相似，只是金属离子与所有三个α-、β-和γ-磷酸配位，但不与蛋白质配位。在反应中转移的γ-磷酸不与蛋白质结合。三元复合物的另一个特征是Arg-38移动到一个位置，其胍基与结合的3-PG的N端结构域、C端结构域和1-羧基形成三重相互作用。尽管在三元复合物中观察到了铰链弯曲构象变化，但它并不比在3-PG二元复合物中观察到的大。为了将两个结合底物之间的距离减小到与已知在PGK中发生的直接直线转移一致的值，我们模拟了蛋白质结构中位于结合底物之间的明显裂缝的闭合。这种闭合暗示了一种催化机制，该机制涉及Arg-38和螺旋14的N端对γ-磷酸的“捕获”，螺旋14具有保守的Gly-Gly-Gly磷酸结合基序。我们提出，3-PG的1-羧基对γ-磷的亲核攻击发生在γ-磷酸被捕获之后，导致一个五配位过渡态，该过渡态可能通过螺旋14 N端的NH基团和Arg-38的胍基提供的氢键而稳定。在反应过程中，金属离子被认为迁移到一个与α-和β-磷酸以及Asp-374的羧基配位的位置。该机制与来自二元和三元底物复合物的结构信息以及许多溶液数据一致，并如定点诱变所示，赋予Arg-38主要的催化作用。
