Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy.

A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.     

A mutant pyruvate dehydrogenase complex of Escherichia coli deleted in the (alanine + proline)-rich region of the acetyltransferase component

The acetyltransferase chains of the pyruvate dehydrogenase complex of Escherichia coli contain conformationally mobile (alanine + proline)-rich segments that link the lipoyl domains to each other and to the subunit-binding and catalytic domain, and facilitate the intramolecular coupling of active sites in the complex. A deletion of 12 of the 32 residues of the (Ala + Pro)-rich segment of an acetyltransferase containing only one lipoyl domain was made by deleting the corresponding segment of the aceF gene. A pyruvate dehydrogenase complex was still produced and the catalytic activity of the restructured complex, including active-site coupling, was not detectably impaired.     

Investigation of the mechanism of active site coupling in the pyruvate dehydrogenase multienzyme complex of Escherichia coli by protein engineering

Site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate a nested set of deletions in the long (alanine + proline)-rich sequence that separates the lipoyl domain from the dihydrolipoamide dehydrogenase-binding domain in the "one-lipoyl domain" dihydrolipoamide acetyltransferase polypeptide chains of a pyruvate dehydrogenase multienzyme complex. The deletions reduced the number of residues in this sequence successively from 32 to 20, 13, 7 and just 1 residue. In all instances, pyruvate dehydrogenase complexes were still assembled in vivo around cores containing the deleted chains, and those with the two shortest deletions were essentially fully active. However, the two most severe deletions caused falls of 50% or more in specific catalytic activity. Similarly, although shortening the interdomain sequence to 20 residues left the system of active-site coupling unimpaired, cutting it to 13 residues or less caused substantial falls in the reductive acetylation of the lipoyl domains and corresponding losses of active-site coupling. The changes in specific catalytic activity and active-site coupling that accompanied the shortening of the (alanine + proline)-rich segment were reflected in the poorer growth rates of the relevant strains of E. coli on stringent substrates. All these results are consistent with this (alanine + proline)-rich sequence acting as a linker region that facilitates the movements of the lipoyl domains required for full catalytic activity and active-site coupling in the complex. The other two such sequences that separate the additional lipoyl domains in the N-terminal half of the wild-type "three-lipoyl domain" dihydrolipoamide acetyltransferase chain are presumed to function similarly. This role is consistent with the conformational flexibility assigned to these segments from previous studies based on 1H nuclear magnetic resonance spectroscopy and protein engineering.     

Site-directed mutagenesis and 1H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle (Mr approximately 4 x 10(6)), sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz 1H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pKa (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is thought to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

Conformational studies of the interdomain linker peptides in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two peptides (PEP1, 26 residues, and PEP2, 22 residues) were synthesized with amino acid sequences identical to two of the long segments of polypeptide chain rich in alanine, proline, and charged amino acids that link the lipoyl domains together in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The circular dichroism and 400-MHz 1H NMR spectra of the peptides indicated that they lacked regular secondary structure. Even in the presence of 45% (v/v) hexafluoroisopropanol, they appeared to acquire a helical content of only 23-25%. However, 13C NMR spectroscopy revealed that the Ala-Pro peptide bonds were all (> 95%) in the trans configuration, compared with a value of 87% for the Ala-Pro bond in the model peptide AAPA, which is a recurrent sequence motif in PEP1 and PEP2. Likewise in peptides representing the N- and C-terminal halves of peptide PEP2, the Ala-Pro bonds were again all (> 95%)-trans, suggesting that peptide length is the essential determinant of the cis:trans ratio. Antisera were raised against peptides PEP2 and PEP3, the latter representing a third interdomain segment of polypeptide chain (Radford, S. E., Laue, E. D., Perham, R. N., Martin, S. R., and Appella, E. (1989a) J. Biol. Chem. 264, 767-775). Despite extensive sequence similarity among peptides PEP1, PEP2, and PEP3, only limited immunological cross-reactivity was observed, which suggests that the antigenic epitope(s) in the peptides are different and distinct. It is likely that these peptides are representative of a class of inter-domain linkers or spacers found in a wide variety of proteins and endowed with varying degrees of flexibility and stiffness to match their particular biological purpose.     

Domain structure and 1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.     

Two lipoyl domains in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis

A fragment of DNA incorporating the gene, pdhC, that encodes the dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis was cloned and a DNA sequence of 2360 bp was determined. The pdhC gene (1620 bp) corresponds to an E2 chain of 539 amino acid residues, Mr 56,466, comprising two lipoyl domains, a peripheral subunit-binding domain and an acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, proline and charged amino acids. The S. faecalis E2 chain differs in the number of its lipoyl domains from the E2 chains of all bacterial pyruvate dehydrogenase complexes hitherto described.     

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.     

Expression in Escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

A sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. Approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on Lys-42 and could be reductively acetylated by the B stearothermophilus enzyme complex. A small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on Lys-42. The 400 MHz 1H NMR spectra of the lipoylated and unlipoylated domains were essentially identical and closely resembled that of the native lipoyl domain.     

Solution structure of the lipoyl domain of the chimeric dihydrolipoyl dehydrogenase P64K from Neisseria meningitidis.

The antigenic P64K protein from the pathogenic bacterium Neisseria meningitidis is found in the outer membrane of the cell, and consists of two parts: an 81-residue N-terminal region and a 482-residue C-terminal region. The amino-acid sequence of the N-terminal region is homologous with the lipoyl domains of the dihydrolipoyl acyltransferase (E2) components, and that of the C-terminal region with the dihydrolipoyl dehydrogenase (E3) components, of 2-oxo acid dehydrogenase multienzyme complexes. The two parts are separated by a long linker region, similar to the linker regions in the E2 chains of 2-oxo acid dehydrogenase complexes, and it is likely this region is conformationally flexible. A subgene encoding the P64K lipoyl domain was created and over-expressed in Escherichia coli. The product was capable of post-translational modification by the lipoate protein ligase but not aberrant modification by the biotin protein ligase of E. coli. The solution structure of the apo-domain was determined by means of heteronuclear NMR spectroscopy and found to be a flattened beta barrel composed of two four-stranded antiparallel beta sheets. The lysine residue that becomes lipoylated is in an exposed beta turn that, from a [1H]-15N heteronuclear Overhauser effect experiment, appears to enjoy substantial local motion. This structure of a lipoyl domain derived from a dihydrolipoyl dehydrogenase resembles that of lipoyl domains normally found as part of the dihydrolipoyl acyltransferase component of 2-oxo acid dehydrogenase complexes and will assist in furthering the understanding of its function in a multienzyme complex and in the membrane-bound P64K protein itself.     

Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14-base-pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS-like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS-like sequence is homologous with other intercistronic sequences including that between the sdhB and sucA genes, the overall gene organisation being: sdhB-IS-sucAsucB-IS-. The patterns of codon usage indicate that sucB may be more strongly expressed than sucA, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex. The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceF gene product) indicates that each contains two analogous domains, an amino-terminal lipoyl domain linked to a carboxy-terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated approximately 100-residue lipoyl binding regions containing two short (approximately 19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one approximately 100-residue lipoyl binding region, with approximately 27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each approximately 300 residues, have an overall homology of 34% and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy-terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline-alanine segment is found approximately 50 residues into the subunit binding domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural requirements within the lipoyl domain for the Ca2+-dependent binding and activation of pyruvate dehydrogenase phosphatase isoform 1 or its catalytic subunit

The inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2) 60-mer forms a Ca(2+)-dependent complex with the pyruvate dehydrogenase phosphatase 1 (PDP1) or its catalytic subunit, PDP1c, in facilitating large enhancements of the activities of PDP1 (10-fold) or PDP1c (6-fold). L2 binding to PDP1 or PDP1c requires the lipoyl-lysine prosthetic group and specificity residues that distinguish L2 from the other lipoyl domains (L1 in E2 and L3 in the E3-binding component). The L2-surface structure contributing to binding was mapped by comparing the capacities of well folded mutant or lipoyl analog-substituted L2 domains to interfere with E2 activation by competitively binding to PDP1 or PDP1c. Our results reveal the critical importance of a regional set of residues near the lipoyl group and of the octanoyl but not the dithiolane ring structure of the lipoyl group. At the other end of the lipoyl domain, substitution of Glu(182) by alanine or glutamine removed L2 binding to PDP1 or PDP1c, and these substitutions for the neighboring Glu(179) also greatly hindered complex formation (E179A > E179Q). Among 11 substitutions in L2 at sites of major surface residue differences between the L1 and L2 domains, only the conversion of Val-Gln(181) located between the critical Glu(179) and Glu(182) to the aligned Ser-Leu sequence of the L1 domain greatly reduced L2 binding. Certain modified L2 altered E2 activation of PDP1 differently than PDP1c, supporting significant impact of the regulatory PDP1r subunit on PDP1 binding to L2. Our results indicate hydrophobic binding via the extended aliphatic structure of the lipoyl group and required adjacent L2 structure anchor PDP1 by acting in concert with an acidic cluster at the other end of the domain.     

Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.     

Interaction of avidin with the lipoyl domains in the pyruvate dehydrogenase multienzyme complex: three-dimensional location and similarity to biotinyl domains in carboxylases.

Avidin can form intermolecular cross-links between particles of the pyruvate dehydrogenase multienzyme complex from various sources. Avidin does this by binding to lipoic acid-containing regions of the dihydrolipoamide acetyltransferase polypeptide chains that comprise the structural core of the complex. It is inferred that the lipoyl domains of the acetyltransferase chain extend outwards from the interior of the enzyme particle, interdigitating between the subunits of the other two enzymes bound peripherally in the assembled structure, with the lipoyl-lysine residues capable of reaching to within at least 1-2 nm of the outer surface of the enzyme complex (diameter ca. 37 nm). The distribution of enzymic activities between different domains of the dihydrolipoamide acetyltransferase chain implies that considerable movement of the lipoyl domains is a feature of the catalytic activity of the enzyme complex. There is evidence that the lipoyl domain of the 2-oxo acid dehydrogenase complexes is similar in structure to a domain that binds the cofactor biotin, also in amide linkage with a specific lysine residue, in the biotin-dependent class of carboxylases.     

Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli

The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex. The structure of the unlipoylated form of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybrid domain determined previously [Green et al. (1995) J. Mol. Biol. 248, 328-343]. The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the sheets; the N- and C-termini lie close together at the opposite end of the molecule in the other beta-sheet. Measurement of (15)N NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the residues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue. This implies that the lipoyl-lysine side chain may not sample the full range of conformational space once thought. Moreover, reductive acetylation of the lipoylated domain (E2plip(holo) --> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonances were observed for several residues. This implies a change in conformation and the existence of multiple conformations of the domain on reductive acetylation, which may be important in stabilizing this catalytic intermediate.     

The Mr-50 000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions

The mammalian pyruvate dehydrogenase complex, Mr 8.5 X 10(6), contains an additional tightly bound 50 000-Mr polypeptide, component X, which copurifies with the intact assembly. Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with 125I-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-14C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These protein-chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70 000) subunit. Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.     

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.     

The pyruvate dehydrogenase multienzyme complex

During the review period, several structures of component enzymes and domains of enzymes of this multienzyme complex were determined. Three structures of the flavoprotein component, dihydrolipoamide dehydrogenase, became available. The structure of the core component, dihydrolipoyl acetyltransferase, can in principle be constructed from the known structures of its modules: the lipoyl, the peripheral subunit-binding and the catalytic domain. Dynamic aspects, such as the structure and function of the inter-domain linkers in dihydrolipoyl acetyltransferase and the conformational changes invlved in the mechanism of electron transfer in dihydrolipoamide dehydrogenase, remain to be clarified. Although several questions concerning the structure of the individual components of the complex have been solved, there is still much to learn about the assembly pathway. In mammalian complexes, the structure and function of protein X remains something of a riddle.     

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.