Studies of the thiamin pyrophosphate binding site of Escherichia coli pyruvate oxidase. Evidence for an essential tryptophan residue.
Explore the source record for details and available documents.
Binding Sites↗
Biomedical subjects
T A O'Brien
Publications and source records attributed to T A O'Brien.
Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases.
A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.
Acetaldehyde↗
Studies on the interaction between Escherichia coli pyruvate oxidase and a detergent activator by utilization of the fluorescence probe bis(8-p-toluidino-1-naphthalenesulfonate).
Explore the source record for details and available documents.
Binding Sites↗
Complex formation between the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and valinomycin in the presence of potassium.
Spectroscopic evidence is presented which indicates that the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the peptide antibiotic valinomycin form a complex in the presence of potassium. Complex formation has been observed both in aqueous and nonaqueous media. Several techniques have been used to indicate the existence of a complex in aqueous solution. In the presence of valinomycin and K+, the absorption spectrum of FCCP is significantly perturbed, and there is also a large induced circular dichroism signal. In addition, the previously characterized complex which forms between valinomycin, K+, and the fluorescent probe 8-anilino-1-naphthalene-sulfonate (ANS) in aqueous solution is apparently disrupted by the addition of FCCP. The result is an effective quenching of the fluorescence due to the bound probe as it is displaced from the valinomycin.K+ by the uncoupler. In a nonpolar solvent, the absorption spectrum of FCCP is also perturbed by valinomycin in the presence of K+, again indicating the formation of a complex. These data point to the importance of considering the role of valinomycin.K+.uncoupler complex in interpreting physiological or ion transport data in which these substances have been used together.
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
Regulation by lipids of cofactor binding to a peripheral membrane enzyme: binding of thiamin pyrophosphate to pyruvate oxidase.
Explore the source record for details and available documents.
Cell Membrane↗
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↗
Evaluation of atypical human immunodeficiency virus immunoblot reactivity in blood donors.
Blood donors reactive by enzyme-linked immunosorbent assay for antibody to the human immunodeficiency virus (HIV) who showed atypical patterns of viral core protein reactivity on Western blot were monitored for several months. Characterization of their antibodies was performed by 1) use of recombinant HIV proteins; 2) determination of cross-reactivity to HTLV-I, HTLV-II, and HTLV-IV: 3) assessment of immune status; and 4) identification of potentially interfering autoantibodies. Nineteen of 20 donors maintained the same HIV antibody reactivity throughout the follow-up period; the other donor became fully antibody-positive. Eighteen of 20 donors' sera showed clear reactivity with HIV recombinant core proteins. Ten of 19 donor samples demonstrated cross-reactivity to HTLV-IV; 3 of these 10 also cross-reacted with HTLV-I. The immune status of all donors was normal, although the medical histories and HLA antibody screens suggested possible autoimmune reactivity in 9 of 18 donors. During follow-up interviews, three donors reported possible risk factors for HIV infection that had not been acknowledged at the time of blood donation. We conclude that exclusion of donors with these atypical serologic test results is warranted while further studies to determine significance are being conducted.
Adult↗