Crystal structures of pyruvate phosphate dikinase from maize revealed an alternative conformation in the swiveling-domain motion

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.     

Pyruvate dehydrogenase from Azotobacter vinelandii. Properties of the N-terminally truncated enzyme.

The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA. Previously, limited proteolysis experiments indicated that the N-terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) [Hengeveld, A. F., Westphal, A. H. & de Kok, A. (1997) Eur J. Biochem. 250, 260-268]. To further investigate this hypothesis N-terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized. Up to nine N-terminal amino acids could be removed from E1p without effecting the properties of the enzyme. Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p. The 48 amino acid deletion mutant (E1pdelta48) is catalytically fully functional: it has a Vmax value identical to that of wild-type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule. In contrast, the S0.5 for pyruvate is decreased. A heterodimer was constructed containing one subunit of wild-type E1p and one subunit of E1pdelta48. From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N-terminal domains are needed for the binding of E1p to E2p. The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N-terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p.     

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.     

Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands

Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316-321, which enclose the nucleotide beta and gamma phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.     

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with f/fo = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa. It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.     

In vivo and in vitro analyses of single-amino acid variants of the Salmonella enterica phosphotransacetylase enzyme provide insights into the function of its N-terminal domain

The function of the N-terminal domain ( approximately 350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mm acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k(cat) 2.5-3-fold > k(cat) Pta(WT). Results from sedimentation equilibrium experiments are consistent with Pta(WT) being a trimer. Pta variants formed more hexamer than the Pta(WT) protein. NADH inhibited Pta(WT) activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta(R252H). Pyruvate stimulated Pta(WT) activity, and its effect was potentiated in the variants, being most pronounced on Pta(R252H).     

Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, pyruvate, and phosphate with AMP, phosphoenolpyruvate (PEP), and pyrophosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis.     

Swiveling domain mechanism in pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

Investigation of the role of the domain linkers in separate site catalysis by Clostridium symbiosum pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible reaction: ATP + P(i) + pyruvate <--> AMP + PP(i) + PEP using Mg2+ and NH4+ ions as cofactors. The reaction takes place in three steps, each mediated by a carrier histidine residue located on the surface of the central domain of this three-domain enzyme: (1) E-His + ATP <--> E-His-PP.AMP, (2) E-His-PP.AMP + P(i) <--> E-His-P + AMP + PP(i), (3) E-His-P + pyruvate <--> E-His + PEP. The first two partial reactions are catalyzed at an active site located on the N-terminal domain, and the third partial reaction is catalyzed at an active site located on the C-terminal domain. For catalytic turnover, the central domain travels from one terminal domain to the other. The goal of this work is to determine whether the two connecting linkers direct the movement of the central domain between active sites during catalytic turnover. The X-ray crystal structure of the enzyme suggests interaction between the two linkers that may result in their coordinated movement. Mutations were made at the linkers for the purpose of disrupting the linker-linker interaction and, hence, synchronized linker movement. Five linker mutants were analyzed. Two of these contain 4-Ala insertions within the solvated region of the linker, and three have 3-residue deletions in this region. The efficiencies of the mutants for catalysis of the complete reaction as well as the E-His + ATP <--> E-His-PP.AMP partial reaction at the N-terminal domain and the E-His + PEP <--> E-His-P + pyruvate reaction at the C-terminal domain were measured to assess linker function. Three linker mutants are highly active catalysts at both active sites, and the fourth is highly active at one site but not the other. These results are interpreted as evidence against coordinated linker movement, and suggest instead that the linkers move independently as the central domain travels between active sites. It is hypothesized that while the linkers play a passive role in central domain-terminal domain docking, their structural design minimizes the conformational space searched in the diffusion process.     

Exceptional characteristics of heterotetrameric (α2β2) E1p of the pyruvate dehydrogenase complex from Zymomonas mobilis: expression from an own promoter and a lipoyl domain in E1β

In the pyruvate dehydrogenase complex (PDHC) of Zymomonas mobilis the beta subunit of the pyruvate dehydrogenase (E1p) as well as the acetyltransferase (E2p) contain an N-terminal lipoyl domain. Both lipoyl domains were acetylated in vitro using 2-14C-pyruvate as a substrate, demonstrating that both lipoyl domains can accept acetyl groups from the E1 component. As previously shown the structural genes (pdhA alpha beta, pdhB, lpd) encoding the pyruvate dehydrogenase complex of Z. mobilis are located in two distinct gene clusters, pdhA alpha beta and pdhB-orf2-lpd (U. Neveling et al. (1998) J. Bacteriol. 180, 1540-1548). Analysis of pdh gene expression using lacZ fusions revealed that the DNA fragments upstream of pdhA alpha, pdhB and lpd each have promoter activities. These pdh promoter activities were 7-30-fold higher in Z. mobilis than in Escherichia coli.     

Structures of the human pyruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal domains

Dihydrolipoyl acetyltransferase (E2) is the central component of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA. Structural comparison by cryo-electron microscopy (cryo-EM) of the human full-length and truncated E2 (tE2) cores revealed flexible linkers emanating from the edges of trimers of the internal catalytic domains. Using the secondary structure constraints revealed in our 8 A cryo-EM reconstruction and the prokaryotic tE2 atomic structure as a template, we derived a pseudo atomic model of human tE2. The active sites are conserved between prokaryotic tE2 and human tE2. However, marked structural differences are apparent in the hairpin domain and in the N-terminal helix connected to the flexible linker. These permutations away from the catalytic center likely impart structures needed to integrate a second component into the inner core and provide a sturdy base for the linker that holds the pyruvate dehydrogenase for access by the E2-bound regulatory kinase/phosphatase components in humans.     

Three dimensional structure of porcine pancreatic alpha-amylase at 2.9 A resolution. Role of calcium in structure and activity.

The crystal structure of porcine pancreatic alpha-amylase (PPA) has been solved at 2.9 A resolution by X-ray crystallographic methods. The enzyme contains three domains. The larger, in the N-terminal part, consists of 330 amino acid residues. This central domain has the typical parallel-stranded alpha-beta barrel structure (alpha beta)8, already found in a number of other enzymes like triose phosphate isomerase and pyruvate kinase. The C-terminal domain forms a distinct globular unit where the chain folds into an eight-stranded antiparallel beta-barrel. The third domain lies between a beta-strand and a alpha-helix of the central domain, in a position similar to those found for domain B in triose phosphate isomerase and pyruvate kinase. It is essentially composed of antiparallel beta-sheets. The active site is located in a cleft within the N-terminal central domain, at the carboxy-end of the beta-strands of the (alpha beta)8 barrel. Binding of various substrate analogues to the enzyme suggests that the amino acid residues involved in the catalytic reaction are a pair of aspartic acids. A number of other residues surround the substrate and seem to participate in its binding via hydrogen bonds and hydrophobic interactions. The 'essential' calcium ion has been located near the active site region and between two domains, each of them providing two calcium ligands. On the basis of sequence comparisons this calcium binding site is suggested to be a common structural feature of all alpha-amylases. It represents a new type of calcium-protein interaction pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: An immunological study using specific anti-phosphorylation site-antibodies

A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C₄-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C₄ PEPCs mutated in the N-terminal phosphorylation domain and a C₃-like PEPC isozyme from Sorghum behaved similarly to their C₄ counterpart with respect to these phosphorylation-site antibodies.Abbreviations PEPC phospho enolpyruvate carboxylase - PKA catalytic subunit of the cAMP-dependent protein kinase - KLH Keyhole Limpet Haemocyanin - IgG immunoglobulin G - PEP phospho enolpyruvate - SDS-PAGE sodium dodecyl sulfate, polyacrylamide gel electrophoresis - MDH malate deshydrogenase     

Chain folding in the dihydrolipoyl acyltransferase components of the 2-oxo-acid dehydrogenase complexes from Escherichia coli. Identification of a segment involved in binding the E3 subunit

The state of assembly of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes was examined after the dihydrolipoyl acyltransferase (E2) component of each enzyme system had been subjected to varying degrees of limited proteolysis. Dissociation of the dihydrolipoyl dehydrogenase (E3) component accompanied specifically the excision of a homologous segment of each E2 chain that connects the N-terminal lipoyl domain(s) with a C-terminal catalytic domain. The latter remains aggregated as a 24-mer and retains its capacity to bind the 2-oxo-acid decarboxylase (E1) component. The relevant segment of the E2o chain from the 2-oxoglutarate dehydrogenase complex was isolated and shown to be a folded protein which still binds to E3.     

The 3.0 A resolution crystal structure of glycosomal pyruvate phosphate dikinase from Trypanosoma brucei

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement. The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.     

Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3.

Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.     

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.     

Probing l-Pyruvate Kinase Regulatory Phosphorylation Site by Mutagenesis

The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR¹⁰AS ¹² VAQLTQEL²⁰GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme??s active site, affected the enzyme??s activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme??s main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure.     

Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.