Specificity Determinants for the Pyruvate Dehydrogenase Component Reaction Mapped with Mutated and Prosthetic Group Modified Lipoyl Domains

Abstract

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys 173 ), with three analogs substituted for the lipoyl group on Lys 173, and with selected L1 mutants. Besides Lys 173 mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 Å on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. The pyruvate dehydrogenase complex (PDC) 1 oxidatively decarboxylates pyruvate with the production of acetyl-CoA and NADH. The PDC reaction results from the sequential catalysis by the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. In the intermediate steps, the lipoyl groups on mobile lipoyl domains of the E2 are reductively acetylated by E1, de-acetylated by E2, and re-oxidized by E3. Thus, lipoyl domains play a vital role in connecting the sequential series of reaction catalyzed by these three components. In mammalian PDC, two lipoyl domains, L1 and L2, and an E1-binding domain are provided in the outer structure of the E2 (1, 2). These flexibly held domains surround a pentagonal dodecahedron shaped inner core formed by association of 60 C-terminal catalytic domains of E2. The capacity of lipoyl domains to rapidly move between active sites is provided by highly flexible interdomain linker segments which are located between the globular domains of E2. In mammalian PDC-E2 these linker regions are 20 to 30 amino acids in length and are high in alanine and proline residues; prolines introduce stiffness in these interdomain segments (3). Mammalian PDC has a third lipoyl domain on an E3-binding protein (E3BP) which has a similar segmented structure (1).In the first step of the E1 reaction, the bound thiamin diphosphate (TPP) forms an adduct with pyruvate which is rapidly decarboxylated to generate 2-(1-hydroxyethylidene)-TPP (HE-TPP) (4). In the second step, reductive acetylation of a lipoyl group is achieved by linking the oxidation of the hydroxyethylidene group to the reduction of the disulfide in the dithiola...

Solution Structure and Dynamics of the Lipoic Acid-bearing Domain of Human Mitochondrial Branched-chain α-Keto Acid Dehydrogenase Complex

“…Subsequent studies showed that the interactions between LBD and E1 are more complex and are not attributable to a single determinant on the LBD domain (64 -66). In fact alanine substitutions of residues corresponding to Ile 11 , Gly 12 , and Gly 14 in L1 loop and Thr 50 and Gln 33 in the far end of the hbLBD structure were shown to have marked reduction in the E1 reaction rate (64). Thus, the interaction site between E1 and LBD appears to encompass surfaces that extend from one end to the other in both structures.…”

Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

“…The absence of the lag-phase for the alanine substitution of site 1 and glutamate substitution of sites 2 or 3 (Table I) may indicate the inability of the phosphorylated E1 to undergo the activation during catalysis. Gong et al (49) suggested that it is not the decarboxylation but interaction of lipoyl domain with E1 that facilitates structural changes within E1 and subsequently the efficient reductive acetylation. The negative charge and the size of the phosphoryl group located in the substrate channel could prevent the interaction of E1 with the negatively charged region (Glu-162, Glu-167) of the lipoyl domain (12).…”

The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes

“…The low K m value of 26 M for the free lipoyl domain indicates that it has appreciable affinity for residues near the active site of E 1 (8). Studies of mutations in the lipoyl domains revealed a specificity loop in which several residues in the lipoyl domain confer specificity on the reductive acylation reaction (29)(30)(31). We have docked the lipoyl domains so that this specificity loop faces the E 1 complex near its active site (Fig.…”