A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase

Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp⁵⁹⁰ and Tyr⁶²⁸ and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes.     

Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs

Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the MgATP- and bicarbonate-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in central metabolism. The carboxyltransferase (CT) domain of PC catalyzes the transfer of a carboxyl group from carboxybiotin to the accepting substrate, pyruvate. It has been hypothesized that the reactive enolpyruvate intermediate is stabilized through a bidentate interaction with the metal ion in the CT domain active site. Whereas bidentate ligands are commonly observed in enzymes catalyzing reactions proceeding through an enolpyruvate intermediate, no bidentate interaction has yet been observed in the CT domain of PC. Here, we report three X-ray crystal structures of the Rhizobium etli PC CT domain with the bound inhibitors oxalate, 3-hydroxypyruvate, and 3-bromopyruvate. Oxalate, a stereoelectronic mimic of the enolpyruvate intermediate, does not interact directly with the metal ion. Instead, oxalate is buried in a pocket formed by several positively charged amino acid residues and the metal ion. Furthermore, both 3-hydroxypyruvate and 3-bromopyruvate, analogs of the reaction product oxaloacetate, bind in an identical manner to oxalate suggesting that the substrate maintains its orientation in the active site throughout catalysis. Together, these structures indicate that the substrates, products and intermediates in the PC-catalyzed reaction are not oriented in the active site as previously assumed. The absence of a bidentate interaction with the active site metal appears to be a unique mechanistic feature among the small group of biotin-dependent enzymes that act on α-keto acid substrates.     

Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.     

Activation and inhibition of pyruvate carboxylase from Rhizobium etli

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.     

In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum

The C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C(4)-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C(3) carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C(4) metabolites for biosynthesis.     

Pyruvate carboxylase is expressed in human skeletal muscle

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable by streptavidin blot and describe the presence of considerable amounts of PC in cultured human myotubes and in human muscle tissue.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.     

The active species of "CO2" utilized in ferredoxin-linked carboxylation reactions.

The active species of "CO2", i.e. CO2 or HCO-3(H2CO3) utilized by enzymes catalyzing ferredoxin-linked carboxylation reactions was determined. The enzyme investigated was pyruvate synthase from Clostridium pasteurianum (EC 1.2.7.1; Pyruvate: ferredoxin oxidoreductase). Data were obtained which were compatible with those expected if CO2 is the active species. The dissociation constant (Ks) of the enzyme-CO2 complex was measured. At pH 7.2 Ks for CO2 of pyruvate synthase was found to be approximately 5 mM.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids.

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.     

Inactivation of <Emphasis Type="Italic">pycA</Emphasis>, encoding pyruvate carboxylase activity,increases poly-β-hydroxybutyrate accumulation in <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> on solid medium

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly--hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene (pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly--hydroxybutyrate.     

Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

Corynebacterium glutamicum possesses high in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed (13)C labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate L-aspartate, alpha-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in vivo fluxes, and metabolite concentrations were exploited to elucidate the in vivo regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.