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2.0 Å resolution structure of a ternary complex of pig muscle phosphoglycerate kinase containing 3‐phospho‐D‐glycerate and the nucleotide Mn adenylylimidodiphosphate
Author(s) -
May Andrew,
Vas Maria,
Harlos Karl,
Blake Colin
Publication year - 1996
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(199603)24:3<292::aid-prot2>3.0.co;2-j
Subject(s) - phosphoglycerate kinase , ternary complex , nucleotide , crystallography , ternary operation , chemistry , stereochemistry , pyrophosphate , enzyme , biochemistry , computer science , programming language , gene
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 β‐ and γ‐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 α‐, β‐, and γ‐phosphates, but not by the protein. The γ‐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 γ‐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 γ‐phosphorus follows the capture of the γ‐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 α‐ and β‐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.