5v9f
Structure of the H477R variant of rat cytosolic PEPCK in complex with beta sulfopyruvate and GTP.
Structural highlights
FunctionPCKGC_RAT Catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Publication Abstract from PubMedMobile Omega-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Omega-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 Omega-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 Omega-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 Omega-loop via the H477R substitution results in the destabilization of the closed state of the Omega-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 kcat 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 Omega-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state. Asymmetric Anchoring Is Required for Efficient Omega-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.,Cui DS, Broom A, Mcleod MJ, Meiering EM, Holyoak T Biochemistry. 2017 Apr 4. doi: 10.1021/acs.biochem.7b00178. PMID:28345895[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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