A Novel Branched-chain Amino Acid Metabolon: PROTEIN-PROTEIN INTERACTIONS IN A SUPRAMOLECULAR COMPLEX

Mohammad Mainul Islam,Reidar Wallin,R Max Wynn,Myra Conway,Hisao Fujii,James A Mobley,David T Chuang,Susan M Hutson

doi:10.1074/jbc.m700198200

Abstract

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.

Highlights

The flow of metabolites through an individual pathway and the integration of multiple metabolic pathways involve a complex interplay of different reactions and regulatory mechanisms
Human human BCATm (hBCATm) Forms a Complex with the Rat Liver Mitochondrial BCKD Complex—A strategy was developed to test whether human recombinant mitochondrial BCATm could form a stable complex with the BCKD complex and/or other mitochondrial proteins using a recombinant hBCATm affinity column to screen for potential interacting proteins
The reduced pyridoxal 5Ј-phosphate (PLP) form of hBCATm was coupled to Sepharose 4B, and a detergent-solubilized liver mitochondrial extract was passed through the column (Fig. 1)

Summary

Introduction

The flow of metabolites through an individual pathway and the integration of multiple metabolic pathways involve a complex interplay of different reactions and regulatory mechanisms. The concept that metabolic enzymes associate to form supramolecular structures was developed over 50 years ago [2], and the term metabolon was introduced by Paul Srere in 1985 [3]. Advantages of such a supramolecular assembly include channeling of substrates between enzymes in a pathway for efficiency, regulating the pathway flux by association and dissociation of relevant enzymes, and targeting the localization of the interacting enzymes with the appropriate intracellular structures [3]. The goal of this study was to determine whether mitochondrial enzymes of the branchedchain amino acid (BCAA) catabolic pathway can associate to form a supramolecular complex. BCATm co-localizes with the second enzyme in the BCAA catabolic pathway, the mitochondrial branchedchain ␣-keto acid dehydrogenase complex (BCKD complex)

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