Investigating Carrier Domain Positioning During Catalytic Turnover in Pyruvate Carboxylase

Abstract

A central debate in protein biochemistry focuses on the mechanism by which ligand binding correlates with protein conformational change. Two primary hypotheses describe this process: the induced‐fit hypothesis states that ligand binding induces a conformational change in the protein, while the conformational ensembles hypothesis states that the protein pre‐exists in multiple conformational states, with ligand binding shifting the equilibrium towards a preferred conformation. Pyruvate carboxylase (PC) is a multi‐domain, swinging‐arm enzyme that coordinates reactions between two remote active sites and serves as a structurally and biophysically accessible paradigm to study the relationship between ligand binding and protein conformational change. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. The biotin cofactor on the biotin carboxyl carrier protein (BCCP) domain is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then translocates to the carboxyltransferase (CT) domain on an opposing subunit, where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate.In order to better understand the mechanism of carrier domain translocation, the equilibrium positioning of the BCCP domain was directly observed using site‐specific cross‐linking to trap the BCCP domain in the putative exo‐binding site on the opposing subunit. This approach permits a detailed analysis of BCCP domain positioning in response to various substrates and allosteric effectors. Samples were first analyzed using SDS‐PAGE and densitometry to measure BCCP cross‐linked dimers as reporters for equilibrium positioning changes. Given that BCCP cross‐linking inactivates the enzyme, rates of inactivation were measured to assess changes in BCCP domain positioning in the presence of substrates and effectors. Finally, intrinsic tryptophan fluorescence changes were measured on fast timescales to more accurately determine the effect of ligands on the rates of BCCP domain repositioning. These studies reveal that substrates and effectors shift the BCCP domain positioning from one domain to another and offer the first evidence that the carrier domain exists in a dynamic equilibrium between active sites.Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.