title: Asymmetric Anchoring Is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.
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-1. 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 kcatfor 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.
authors.affiliation: Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
authors.forename: Danica S
authors.initials: DS
authors.lastname: Cui
authors.affiliation: Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
authors.forename: Aron
authors.initials: A
authors.lastname: Broom
authors.affiliation: Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Mcleod
authors.affiliation: Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Meiering
authors.affiliation: Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
authors.forename: Todd
authors.identifier: 0000-0002-7329-1115
authors.initials: T
authors.lastname: Holyoak
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D010729:Phosphoenolpyruvate Carboxykinase (GTP)