Production of recombinant E1 component of branched-chain α-keto acid dehydrogenase complex

Abstract

The activity state of mitochondrial branched-chain α-keto acid dehydrogenase (BCKDH) is highly regulated according to the physiological requirements for protein synthesis or degradation of excess branched-chain amino acids.1 This regulation is primarily exerted through reversible phosphorylation of the catalytic subunit of BCKDH by a specific protein kinase and a specific phosphoprotein phosphatase. Two phosphorylation sites exist on the E1α subunit although regulation of BCKDH activity results exclusively from phosphorylation of site 1, Ser-293 in rat E1α. Phosphorylation of site 2, Ser-303 in rat E1α, occurs at a significantly slower rate2 and is silent with respect to regulation of BCKDH activity.3 Thiamine pyrophosphate (TPP) and branched-chain α-keto acids (cofactor and substrates for the BCKDH-catalyzed reaction) inhibit the phosphorylation by BCKDH kinase, and it has been suggested that BCKDH kinase inactivates BCKDH E1 by placing a covalently linked phosphate directly into the active site of the dehydrogenase.4 Studies of the mechanism of inactivation of BCKDH by phosphorylation require a ready source of purified subunits of this multienzyme complex that can be reconstituted into an active BCKDH complex under various conditions. To this end we have expressed the BCKDH E1 component as a recombinant enzyme5 in a form that can be easily purified and reconstituted with the purified transacylase core (E2) of the BCKDH complex. The purified recombinant E1 subunit is enzymatically active when combined with E2, can be inactivated by treatment with the purified recombinant BCKDH kinase and ATP, and can be manipulated by site-directed mutagenesis. Materials R408 and VCS M13 are purchased from Stratagene (La Jolla, CA). Vectors pET15 and pET21 are purchased from Novagen (Madison, WI). Nickel-NTA-agarose is purchased from Qiagen (Valencia, CA). Restriction enzymes, restriction enzyme buffers, and other DNA-modifying enzymes are purchased from BRL (Gaithersburg, MD). All chemical reagents for enzyme assay are purchased from Sigma (St. Louis, MO). The site-directed mutagenesis system is purchased from Amersham (Arlington Heights, IL). Native rat liver BCKDH E2 subunit is prepared by a modification of the method of Cook et al.6 according to Shimomura et al.7 The pGroESL plasmid was a kind gift of A. Gatenby (Central Research and Development, Du Pont, Wilmington, DE).