2.0 A resolution structure of a ternary complex of pig muscle phosphoglycerate kinase containing 3-phospho-D-glycerate and the nucleotide Mn adenylylimidodiphosphate.
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.
May
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A
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Vas
M
M
Harlos
K
K
Blake
C
C
D000266:Adenylyl Imidodiphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D005988:Glyceric Acids / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
D005988:Glyceric Acids; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase