5V9F

Structure of the H477R variant of rat cytosolic PEPCK in complex with beta sulfopyruvate and GTP.

Summary for 5V9F

• Entry DOI: 10.2210/pdb5v9f/pdb
• Related: 5V95 5V97 5V9G 5V9H
• Descriptor: Phosphoenolpyruvate carboxykinase, cytosolic [GTP], MANGANESE (II) ION, SODIUM ION, ... (6 entities in total)
• Functional Keywords: phosphoenolpyruvate carboxykinase, lyase
• Biological source: Rattus norvegicus (Rat)
• Total number of polymer chains: 1
• Total formula weight: 70397.82

Authors

Holyoak, T.,Cui, D.S. (deposition date: 2017-03-23, release date: 2017-04-12, Last modification date: 2023-10-04)

Primary citation

Cui, D.S.,Broom, A.,Mcleod, M.J.,Meiering, E.M.,Holyoak, T.
Asymmetric Anchoring Is Required for Efficient Omega-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.
Biochemistry, 56:2106-2115, 2017

PubMed Abstract: Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in k for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state.

PubMed: 28345895
DOI: 10.1021/acs.biochem.7b00178

Experimental method

X-RAY DIFFRACTION (2.05 Å)