Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system. 1. Biochemical studies.
Lipoamide dehydrogenase or dihydrolipoamide dehydrogenase (EC 1.8.1. 4) is the E3-protein component of the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. It is also the L-protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2-oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H-protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2-carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H-protein during the course of the reaction catalysed by the L-protein. This provided a valuable new tool with which to study the catalytic properties of the lipoamide dehydrogenase. The L-protein displayed a much higher affinity for the dihydrolipoyl H-protein than for free dihydrolipoamide. The oxidation of the dihydrolipoyl H-protein was not affected by the presence of structurally related analogues (apoH-protein or octanoylated H-protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P-protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H-protein, containing the lipoamide moiety, displayed Km values for the L-protein close to that found for the H-protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P-protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H-protein. From our results we concluded that no structural interaction is required for the L-protein to catalyse the oxidation of the dihydrolipoyl H-protein. We discuss the possibility that one function of the H-protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the lipoamide dehydrogenase.
Neuburger
M
M
Laboratoire de Physiologie Cellulaire Végétale, CEA/CNRS/Université Joseph Fourier, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Polidori
A M
AM
Piètre
E
E
Faure
M
M
Jourdain
A
A
Bourguignon
J
J
Pucci
B
B
Douce
R
R
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D006023:Glycoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D018532:Pisum sativum / Q000378:metabolism; D010720:Phosphines / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D053482:beta 2-Glycoprotein I
D000594:Amino Acid Oxidoreductases; D002352:Carrier Proteins; D008058:Dihydrolipoamide Dehydrogenase; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D006023:Glycoproteins; D007202:Indicators and Reagents; D010720:Phosphines; D010940:Plant Proteins; D011994:Recombinant Proteins; D008063:Thioctic Acid; D053482:beta 2-Glycoprotein I; C080938:tris(2-carboxyethyl)phosphine; C013091:lipoamide