Synergistic effects of substrate induced conformational changes in phosphoglycerate kinase activation.

Presenter's Summary

Phosphoglycerate kinase (PGK) catalyzes one of the key steps in glycolysis:

The main question arised from previous published PGK crystal structures of various species is that the bound substrates are too far apart for catalysis. This leads to the hypothesis that PGK is a hinge-bending enzyme. This paper presents the 2.8 Å ternary structure of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. The structure adopts the catalytic conformation which allows an atomic understanding of how this hinge-bending motion is accomplished in PGK.

PGK is a monomeric enzyme consisting of N-terminal and C-terminal domains linked by a highly conserved hinge region. The ternary complex shows a dramatic closing (about 6 Å closer compared to the other reported PGK structures) of the large cleft between two domains. The large conformational change brings the two substrates in close proximity; they are now well-aligned for catalysis. Figure 2 in the paper shows this cleft closing movement.

The cleft closing mechanism is proposed in the paper as follows:

• 3-PGA binds to the Arg-rich basic patch in the N-terminus which induces a conformational change of the 3-PGA binding loop.
• The conformational change in the binding loop forces helix 14 to translate (Fig.5), resulting in an 8 degree hinge bend. (see Fig.2)
• After the translation, helix 14 is in position to interact with the C-terminal domain once MgADP binds to its site.
• The binding of MgADP induces subtle rearrangement of resdues at the N-terminus of helix 13.
• The combined conformational changes brought upon by the binding of 3-PGA and MgADP enables the formation of a beta-sheet composed of residues preceding helices 13 and 14. This reorganization fixes the helices an dbound substrates in the catalytic conformation.

The authors modeled 1,2 BPA in place of 3-PGA into the PGK active site and showed that the transferring phosphate group on 1,3 BPA is only 4 Å away from the beta-phosphate oxygen nucleophile in MgADP. The transition state proposed involved a pentacoordinated phosphoryl group. One of the three negative oxygens interacts with Mg2+. The authors suggest that the divalent cation bridges the beta-phosphate of ADP and 1-phosphate of 1,3-BPG during catalysis. The second oxygen is stabilized by Arg39. Finally, the third oxygen interacts with the amide of Gly399 at the N-terminus of helix 14.

Why is this paper important?

• This paper demonstrates a special mechanism in which small substrate induced conformational changes combine to produce major conformational changes, arranging substrates in proper orientation for catalysis. It is critical that the major conformational changes are induced only when both substrates are present in the activesite to prevent futile hydrolysis of 1,3 BPG or ATP.
• The findings in this paper can aid in the design of inhibitors which can be developed into antitrypanosomal drugs. The blood stream form of the sleeping sickness parasite is entirely dependent on glycolysis for energy source. Since PGK is the key enzyme in glycolysis, it is an ideal target for the disease.

Abstract of the authors

Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 Å crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.