Measuring the Positioning and Translocation of the Swinging‐Arm Domain of Pyruvate Carboxylase

Abstract

A central goal of studies on large, multi‐domain enzymes such as polyketide synthases and fatty acid synthase is to reveal the mechanism(s) contributing to carrier domain translocation and coordination. Pyruvate carboxylase (PC) is a multi‐domain enzyme with a swinging‐arm domain that coordinates reactions between two remote active sites and can serve as a structurally and biophysically accessible paradigm to study multicomponent swinging‐domain enzymes. PC catalyzes the ATP‐dependent carboxylation of pyruvate to oxaloacetate, using bicarbonate as the carboxyl group donor. This serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. The biotin cofactor on the biotin carrier domain (BCCP) is carboxylated via a MgATP‐dependent reaction in the biotin carboxylase (BC) domain. The BCCP domain then swings to the carboxyltransferase (CT) domain where the carboxyl group is transferred from the biotin cofactor to pyruvate, generating oxaloacetate. These reactions, which take place in distantly located active sites, are tightly coordinated under optimal reaction conditions. In order to better understand the mechanism of coordination, the equilibrium positioning and translocation of the swinging BCCP domain was directly observed using site‐specific cross‐linking and fluorescence anisotropy. Probes were positioned at the CT active site and the so‐called “exo‐binding site”, permitting a detailed analysis of BCCP domain positioning and movement in response to various substrates and allosteric effectors. These studies have revealed that substrates and effectors shift the BCCP domain binding equilibrium 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 is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01.