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Biochemistry. 2017 Apr 18;56(15):2106-2115. doi: 10.1021/acs.biochem.7b00178. Epub 2017 Apr 4.

Asymmetric Anchoring Is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase.

Author information

1
Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.

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 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 Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state.

PMID:
28345895
DOI:
10.1021/acs.biochem.7b00178
[Indexed for MEDLINE]

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