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At least 19 recordsLinked to original sources
PubMed1982

Conformational studies of Escherichia coli pyruvate oxidase.

Pyruvate oxidase (pyruvate:oxygen oxidoreductase (phosphorylating), EC 1.2.3.3) is a peripheral membrane enzyme from Escherichia coli which utilizes the cofactors thiamin pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) to catalyze the decarboxylation of pyruvate to acetic acid and carbon dioxide. The specific activity of the oxidase is enhanced 25-fold when assayed in the presence of certain lipids and detergents. Previous studies have demonstrated that the affinity of pyruvate oxidase for phospholipids and detergents is substantially increased when the flavin is reduced. In this paper, several techniques are utilized to probe both the nature of the active site and the conformational changes in the protein which are concomitant with flavin reduction and with the binding of lipids to the enzyme. Analysis of the circular dichroism spectrum in the far ultraviolet region indicates that neither the binding of lipid activators to the oxidase nor reduction of the enzyme-bound flavin by pyruvate has a significant effect on the average secondary structure of the enzyme. High-resolution electron microscopy demonstrates that at low enzyme concentrations, i.e., assay conditions, incubation of the reduced flavoprotein in the presence of an amphiphilic activator does not alter the quaternary structure of pyruvate oxidase. The results indicate that the conformational changes in the protein due either to reduction of the flavin or to the binding of lipid activators are localized.

Circular Dichroism↗
PubMed1986

Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase.

Pyruvate oxidase from Escherichia coli is a peripheral membrane associated enzyme which is activated by lipids. We have investigated the high-affinity lipid binding site associated with lipid activation of pyruvate oxidase by covalent attachment of [14C]lauric acid to the enzyme. Lauric acid is bound stoichiometrically (1 mol/mol of active sites), and the enzyme is essentially irreversibly activated. Mild tryptic digestion of the modified enzyme shows that the lauric acid is bound within the last 100 residues of the 572-residue monomer. Digestion with thermolysin releases two closely related peptides, A and B, in approximately equal amounts. Comparison of the amino acid composition of peptide A with the entire sequence of the protein shows that peptide A corresponds to the sequence from Ala-543 to Ile-554. The analysis of peptide B is very similar to that of A. Limited sequence analysis of peptide B shows that residue 1 is Ala and residue 2 is labeled. These results support the assignment of residue 1 in peptide B as Ala-543 and indicate that lauric acid is bound to Lys-544. Previous work in this laboratory has shown that pyruvate oxidase may be activated independently of lipids by mild protease digestion. Proteolytic activation is accompanied by the release of a small peptide (residues 550-572) from the carboxyl terminus of the protein. The present work locates the lipid binding site very close to this peptide. The significance of these results for the mechanism of activation of pyruvate oxidase and other lipid-activated systems is discussed.

Amino Acid Sequence↗
PubMed1987

A single-step large-scale purification of pyruvate oxidase.

Pyruvate oxidase is an Escherichia coli peripheral membrane flavoprotein which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. Pyruvate oxidase, like several other peripheral membrane enzymes, can be activated either by binding to lipid amphiphiles or by limited protease digestion. This paper reports a rapid and convenient method for effecting the large-scale purification of pyruvate oxidase from crude enzyme preparations using a Triton X-114 phase separation technique. It appears likely that this purification procedure can be used successfully with the family of enzymes which respond to both lipid and protease activation.

Detergents↗
PubMed1984

Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase.

Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.

Cell Membrane↗
PubMed1980

Studies on the quaternary structure of Escherichia coli pyruvate oxidase.

Pyruvate oxidase is a peripheral membrane enzyme isolated from Escherichia coli. The enzyme catalyzes the oxidative decarboxylation of pyruvate to yield acetate plus CO2. The specific activity of the purified oxidase is stimulated 25-fold by lipids, and this lipid requirement has been the subject of previous studies. Since the enzyme is a tetramer at high protein concentrations (1 mg/ml) and is known to self-aggregate under certain conditions, the question arose as to whether the lipid stimulation observed in the steady state assay might be due to a change in the quaternary structure of the protein, either a dissociation or further association. This report is directed at determining the state of association of pyruvate oxidase under assay conditions by using fluorescence polarization. A photoreactive, nonspecific probe, 1-azidonaphthalene 5-sulfonate, was used to label the protein surface with an extrinsic fluorophore. It is concluded that under steady state assay conditions the oxidase remains tetrameric.

Azides↗
PubMed1993

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase.

BACKGROUND: The crystal structures of three thiamin diphosphate-dependent enzymes that catalyze distinct reactions in basic metabolic pathways are known. These enzymes--transketolase, pyruvate oxidase and pyruvate decarboxylase--also require metal ions such as Ca2+ and Mg2+ as cofactors and have little overall sequence similarity. Here, the crystal structures of these three enzymes are compared. RESULTS: The three enzymes share a similar pattern of binding of thiamin diphosphate and the metal ion cofactors. The enzymes function as multisubunit proteins, with each polypeptide chain folded into three alpha/beta domains. Two of these domains are involved in binding of the thiamin diphosphate and the metal ion. These domains have the same topology of six parallel beta-strands and surrounding alpha-helices. The thiamin diphosphate is bound in a cleft, formed by two domains from two different subunits. Only a few residues are conserved in all three enzymes and these are responsible for proper binding of the cofactors. CONCLUSIONS: Despite considerable differences in quaternary structure and lack of overall sequence homology, thiamin diphosphate binds to the three enzymes in a very similar fashion, and a general thiamin-binding fold can be revealed.

Amino Acid Sequence↗
PubMed1995

Use of pyruvate oxidase to overcome pyruvate inhibition during the lactate to pyruvate reaction for assaying lactate dehydrogenase in serum.

Automated assays of lactate dehydrogenase (LD) in serum are based on measuring the rate of NADH produced in a reverse LD reaction using lactate and NAD. The observed nonlinearity of LD reaction used in earlier assays performed in phosphate buffers has generally been attributed to the formation of a ternary complex of NAD, pyruvate, and phosphate. this is not satisfactory to explain the course of assay reaction carried out in organic buffers. Investigation of the possible causes of nonlinearity during the course of the reverse LD reaction during LD assays performed in Tris or other organic buffers indicated that inhibition of LD activity by pyruvate may be chiefly responsible for the observed effects, especially in serum exhibiting abnormally high LD enzyme activity. Most of the LD activity in serum was inhibited by 5 mMoles/L pyruvate. By contrast, the LD isoenzyme activities were inhibited partially at 0.5 mMole/L pyruvate, LD1 being the most and LD4 the least susceptible. In assays of serum samples with abnormally high LD and PYR concentration using LD reagent containing Tris buffer, pH 9.3, the inclusion of a bacterial pyruvate oxidase (PO) enabled the removal of pyruvate accumulating in situ, making it possible to assay LD activity in the absence of inhibitory concentration of pyruvate. The inclusion of 10 U/L of PO in our routine LD reagent was sufficient to overcome pyruvate inhibition, thus permitting the assay of serum exhibiting high LD activity, hence the extension of the upper limits of linearity of LD assay without compromising assay performance.

Coloring Agents↗
PubMed1980

Specific ligand enhancement of the affinity of E. coli pyruvate oxidase for dipalmitoyl phosphatidylcholine.

Pyruvate oxidase (pyruvate: ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme isolated from Escherichia coli. The enzyme catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2, and is coupled to the E. coli electron traansport chain. In vitro, pyruvate oxidase activity is measured spectrophotometrically using ferricyanide as an electron acceptor. In the presence of dipalmitoyl phosphatidylcholine or a number of other phospholipids, or detergents, the enzymatic specific activity is enhanced about 25-fold. In this paper the interaction between pyruvate oxidase and dipalmitoyl phosphatidylcholine is examined. It is demonstrated that the presence of the ligands involved in catalysis has a substantial influence on the affinity between pyruvate oxidase and dipalmitoyl phosphatidylcholine. In the absence of the substrate (pyruvate) and cofactor (thiamin pyrophosphate) there is no detectable complex formation. However, when both ligands are present, a condition which results in the reduction of the flavoprotein, the interaction between the protein and phospholipid is greatly enhanced. It is clearly shown that the protein-lipid interaction is dramatically modulated by the ligands bound at the catalytic active site on the enzyme and/or by the oxidation-reduction state of the flavin.

Centrifugation, Density Gradient↗
PubMed1980

The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase.

E. coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity. In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain. In vitro, the purified oxidase requires lipids for full enzymatic activity. Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site. The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate. The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein. In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase. The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem. The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence. Quenching titrations are used to obtain the binding isotherm. AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme. This is the concentration range where DNS is an effective activator of the enzyme. This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase. At the DNS concentration which is optimum for activation approx. 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate. The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer. Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide. However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS.

Enzyme Activation↗
PubMed1985

Characterization of the alpha-peptide released upon protease activation of pyruvate oxidase.

The pyruvate oxidase of Escherichia coli is a homo-tetrameric enzyme which can be activated greater than 500-fold (kcat/Km) by limited proteolytic digestion with alpha-chymotrypsin in the presence of pyruvate and thiamine pyrophosphate. The cleavage produces an Mr 2000 peptide (the alpha-peptide) from each subunit and mimics the physiologically important activation of the enzyme by phospholipids. Moreover, the proteolytic cleavage results in the loss of the high affinity lipid-binding site of the enzyme. We now report the isolation and characterization of the alpha-peptide fragment which is cleaved from the carboxyl terminus of each subunit by protease activation. Both the site of cleavage and the sequence of the alpha-peptide have been determined by a combination of Edman degradation of the purified peptide and DNA sequence analysis of the gene encoding the oxidase. The cleavage site lies within a sequence of hydrophobic amino acids predicted to form a beta-sheet. Another segment of the alpha-peptide is predicted to form an amphipathic alpha-helix. Quantitative assessment of the amphipathic nature of this alpha-helix (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623) gives a value very similar to the values for several helical peptides which spontaneously bind to the surface of phospholipid vesicles. From these analyses, we propose that the alpha-peptide may play a role in binding pyruvate oxidase to cell membrane phospholipids in vivo.

Amino Acid Sequence↗
PubMed1982

Role of the divalent metal cation in the pyruvate oxidase reaction.

Purified pyruvate oxidase requires a divalent metal cation for enzymatic activity. The function of the divalent metal cation was studied for unactivated, dodecyl sulfate-activated, and phosphatidylglycerol-activated oxidase. Assays performed in the presence of Mg2+, CA2+, Zn2+, Mn2+, Ba2+, Ni2+, Co2+, Cu2+, and Cr3+ in each of four different buffers, phosphate, 1,4-piperazinediethanesulfonic acid, imidazole, and citrate, indicate that any of these metal cations will fulfill the pyruvate oxidase requirement. Extensive steady state kinetics data were obtained with both Mg2+ and Mn2+. All the data are consistent with the proposition that the only role of the metal is to bind to the cofactor thiamin pyrophosphate (TPP) and that it is the Me2+-TPP complex which is the true cofactor. Values of the Mg2+ and Mn2+ dissociation constants with TPP were determined by EPR spectroscopy and these data were used to calculate the Michaelis constant for the Me2+-TPP complexes. The results show that the Michaelis constants for the Me2+-TPP complexes are independent of the metal cation in the complex. Fluorescence quenching experiments show that the Michaelis constant is equal to the dissociation constant of the Mn2+-TPP complex with the enzyme. It was also shown that Mn2+ will only bind to the enzyme in the presence of TPP and that one Mn2+ binds per subunit. Steady state kinetics experiments with Mn2+ were more complicated than those obtained with Mg2+ because of the formation of an abortive Mn2+-pyruvate complex. Both EPR and steady state kinetics data indicated complex formation with a dissociation constant of about 70 mM.

Buffers↗
PubMed1976

Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column.

An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogenous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.

Chromatography, Affinity↗
PubMed1984

Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum.

Pyruvate oxidase (EC 1.2.3.3) was isolated and characterized from Lactobacillus plantarum. The enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. This pyruvate oxidase is a flavoprotein, with the relatively tightly bound cofactors flavin adenine dinucleotide, thiamine pyrophosphate, and a divalent metal ion, with Mn2+ being the most effective. The enzyme is only slightly inhibited by EDTA, implying that the enzyme-bound metal ion is poorly accessible to EDTA. Only under relatively drastic conditions, such as acid ammonium sulfate precipitation, could a colorless and entirely inactive apoenzyme be obtained. A partial reactivation of the enzyme was only possible by the combined addition of flavin adenine dinucleotide, thiamine pyrophosphate, and MnSO4. The enzyme has a molecular weight of ca. 260,000 and consists of four subunits with apparently identical molecular weights of 68,000. For catalytic activity the optimum pH is 5.7, and the optimum temperature is 30 degrees C. The Km values for pyruvate, phosphate, and arsenate are 0.4, 2.3, and 1.2 mM, respectively. The substrate specificity revealed that the enzyme reacts also with certain aldehydes and that phosphate can be replaced by arsenate. In addition to oxygen, several artificial compounds can function as electron acceptors.

Hydrogen-Ion Concentration↗
PubMed1995

Detection by site-specific disulfide cross-linking of a conformational change in binding of Escherichia coli pyruvate oxidase to lipid bilayers.

Escherichia coli pyruvate oxidase, a peripheral membrane homotetrameric flavoprotein, exposes its C-terminal lipid binding site in the presence of substrate pyruvate and co-factor thiamine pyrophosphate Mg2+ and binds tightly to phospholipid bilayers during catalysis. Using site-specific disulfide cross-linking, we demonstrate that disulfide cross-links are formed between C termini of D560C pyruvate oxidase and that the degree of cross-linking is greatly increased by the presence of substrate and co-factors indicating a conformational change that results in juxtaposition of two subunit C termini. The cross-linked oxidase is enzymatically active and remains able to associate with lipid micelles. These results argue strongly that lipid bilayer binding of pyruvate oxidase involves pairing of the C termini of two subunits.

Amino Acid Sequence↗
PubMed1977

Quasi-elastic light scattering studies on pyruvate oxidase.

Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.). Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase. Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation. In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A. The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.

Centrifugation, Density Gradient↗
PubMed1991

Minimum requirements for protease activation of flavin pyruvate oxidase.

Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.

Amino Acid Sequence↗
PubMed1975

Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids.

The pyruvate oxidase system of Escherichia coli is composed of a soluble flavoprotein, pyruvate oxidase (EC 1.2.2.2, pyruvate:ferricytochrome b1 oxidoreductase), and an electron transport system associated with the cell envelope-membrane fraction. The membrane particles contain 15% lipid by weight. Fractionation of the lipids revealed that abut one-third are neutral lipids and two-thirds are phospholipids. The relative ratio of ubiquinone to menaquinone within the neutral lipid fraction is 15:1 on a molar basis. Removal of the lipids from the membrane particles by extraction with aqueous acetone or hydrolysis of the phospholipids by treatment with Bacillus cereus phospholipase C results in a complete loss of electron transport activity. Analysis of the particles extracted with aqueous acetone revealed that practically all the neutral lipids and 65% of the phospholipids are removed by this treatment. Phospholipase treatment results in a loss of 75% of the membrane phospholipid phosphorus; however, the diglycerides and the neutral lipids produced by phospholipase hydrolysis remain associated with the particles. Addition of neutral lipid and a detergent, hepta-DL-alanyl dodecylamide to the acetone-extracted material results in a restoration of 37% of the original particle activity. Addition of neutral lipid and hepta-DL-alanyl dodecylamide to phospholipase-treated particles completely restores the original electron transport activity. Furthermore, addition of ubiquinone from either yeast (UQ6) or E. coli (UQ8) will restore pyruvate oxidase activity when the quinones are supplemented with photoinactivated neutral lipid. No restoration of activity to phospholipase-treated particles is noted upon the addition of either menaquinone 6 or menaquinone 8 to the reconstitution system. In fact, these compounds appear to suppress restoration of activity when they are added to reaction mixtures containing neutral lipid and phospholipase-treated particles.

Cell Membrane↗