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D E Edmondson

Publications and source records attributed to D E Edmondson.

At least 73 records · Page 4Linked to original sources
PubMed1985

Proton nuclear magnetic resonance studies of 8 alpha-N-imidazolylriboflavin in its oxidized and reduced forms.

The oxidized and hydroquinone forms of synthetic 8 alpha-N-imidazolylriboflavin have been investigated by proton nuclear magnetic resonance spectroscopy at 360 MHz. Proton resonances due to the imidazole ring, isoalloxazine ring, and ribityl side chain have been assigned on the basis of two-dimensional 1H-1H correlated spectra (COSY), selective decoupling, and nuclear Overhauser effect difference spectra and by comparison of computer-simulated with experimental spectra. The effect of pH on the imidazolyl resonances shows a pKa for the unsubstituted imidazole nitrogen of 6.0 +/- 0.1 for the oxidized form and a value of 7.0 +/- 0.1 for the reduced form, in good agreement with the values obtained from oxidation-reduction potential data in a previous paper [Williamson, G., & Edmondson, D. E. (1985) Biochemistry 24, 7790-7797]. Slow exchange of the flavin 8 alpha-methylene and imidazolyl C(2) protons was observed at pH 6.1 but not at pH values below 4.0 for the oxidized form of the flavin. The reduced form, but not the oxidized form, of the flavin exhibits geminal coupling of the 8 alpha-methylene protons and of the C(1') methylene protons of the ribityl side chain. The magnetic nonequivalence of the protons of these two methylene groups is suggested to result from intermolecular association of the reduced flavin in aqueous solutions at the concentrations required for the spectral experiments.

Computers↗
PubMed1985

Effect of pH on oxidation-reduction potentials of 8 alpha-N-imidazole-substituted flavins.

The pKa values for the various ionic forms of 8 alpha-N-imidazolylriboflavin were determined in its oxidized and hydroquinone forms and estimated for its semiquinone form. The pH dependence of the absorption and fluorescence spectral properties and potentiometric titration data show the pKa values for the oxidized form to be 6.02 +/- 0.03 for the 8 alpha-imidazole nitrogen and 9.67 +/- 0.05 for the N(3) position of the flavin ring. The pH dependence of the oxidation-reduction potential was determined by spectrocoulometric titrations, and the data points were compared with computer-simulated plots. Two pKa values for the hydroquinone form of the flavin were determined and assigned. The pKa for the imidazole ring is found to be 6.9 +/- 0.1 and for the N(1) position of the flavin hydroquinone is found to be 5.5 +/- 0.1. Analysis of the pH dependence of the one-electron couples E2 (flavoquinone/flavin semiquinone, Flox/Fl.) and E1 (flavin semiquinone/flavin hydroquinone, Fl./Flred) resulted in an estimated pKa of 6.5 for the 8 alpha-imidazole ring in the flavin semiquinone form. These data show the possible involvement of the ionization of the 8 alpha-imidazole substituent in the redox chemistry of flavoenzymes containing either an 8 alpha-N1- or an 8 alpha-N3-histidyl-linked covalent flavin coenzyme. Future work on oxidation-reduction potentials of this class of enzymes must take into consideration the influence of the 8 alpha-histidyl substituent.

Chemical Phenomena↗
PubMed1985

Evidence for an aldehyde intermediate in the catalytic mechanism of thiamine oxidase.

Thiamine oxidase catalyzes the four-electron oxidation of the 5-hydroxyethyl group of thiamine to form thiamine acetic acid via an aldehyde intermediate. Evidence for the formation of this intermediate is derived from a number of kinetic approaches. The rate of thiamine acetic acid formation, as monitored by the rate of proton release, is subject to substrate inhibition and to inhibition by the presence of semicarbazide while the rate of O2 consumption (due to thiamine oxidation to the aldehyde and subsequently to the carboxylic acid) is unaffected. The transient formation of an intermediate with a maximal absorption at 370 nm in stopped-flow turnover experiments is dependent on the pH and the substrate concentration, and is prevented by the presence of semicarbazide, thus suggesting this transient absorption intermediate to be a result of formation of the aldehyde intermediate. A similar spectral intermediate is observed when hydroxythiamine is the substrate but is not observed with pyrithiamine. In the presence of large concentrations of pyrithiamine, the enzyme undergoes an irreversible inactivation which is not reversed on removal of pyrithiamine or its oxidation products by gel filtration or dialysis. This inhibition is prevented by the presence of thiols or of semicarbazide and is suggested to be due to the release of the aldehyde form of pyrithiamine from the catalytic site, which then reacts with the enzyme in a nonspecific manner. The structure of the 370-nm-absorbing intermediate is currently unknown but is suggested not to be the "yellow form" of thiamine. This suggestion is due to observed differences in absorption spectral properties and to the fact that it can also be formed from hydroxythiamine, which does not form the "yellow form" of thiamine on alkaline treatment. Taken together, these data suggest that, at or below saturating concentrations, thiamine remains bound to the catalytic site during the two sequential two-electron transfer steps, with 2 mol O2 being reduced to 2 mol H2O2. At high concentrations (greater than 10 Km), the intermediate thiamine aldehyde can be displaced from the catalytic site by thiamine simply by a mass-action effect.

Acetates↗
PubMed1985

Electron-spin-resonance studies on flavoenzymes.

Our current knowledge on the spin distribution of flavoenzyme semiquinones would suggest that it is very similar to that determined for model flavin systems. It remains for future work to establish whether this conclusion is valid for a wide range of flavoenzymes as well as for those in which the flavin has become covalently attached to the protein via the 8 alpha position. The improvement in e.s.r. instrumental capabilities, advances in ENDOR spectroscopy, and the application of other techniques such as electron spin echo spectroscopy should result in considerable new information regarding the structures and environment of flavoenzyme semiquinones. Further application of these physical approaches to flavoenzymes in which chemically modified flavins have been substituted for the 'normal' coenzyme (Massey & Hemmerich, 1980) may be particularly informative.

Animals↗
PubMed1985

N-nitrosodialkylamines do not function as substrates for liver monoamine oxidase.

Since reports in the literature [Rowland, I.R., Lake, B. G. & Gangolli, S. D. (1980) Mutat. Res. 72, 63-72] have implicated hepatic monoamine oxidase (EC 1.4.3.4) in the activation of N-nitrosodialkylamines to mutagens, it is of basic pharmacological and biochemical interest to determine if these compounds could serve as substrates for this enzyme. The dialkylnitrosamines N-nitrosodiethylamine, N-nitrosodimethylamine, and N-nitrosodibenzylamine were tested and found not to be substrates, competitive inhibitors, or irreversible inhibitors of liver monoamine oxidase. Thus, any role for monoamine oxidase participation in the mutagenic activation of these compounds must be subsequent to an initial conversion of these compounds to their respective secondary amines.

Animals↗
PubMed1984

31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase.

In addition to the phosphate residues contained in the acid-dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active-center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis has identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH-dependent chemical shift of the phosphorus residue in the molybdenum cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show phosphorus resonances at about -3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at +8.8 ppm and at +13.5 ppm are assigned to those of the pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T.L., Edmondson, D.E., and Husain, M. (1981) Biochemistry 20, 617] and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about -3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50-60% functional enzyme, but increases dramatically in intensity in the desulpho enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line-broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the approximately equal to -3-ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) 'inhibited' form of the enzyme with ethylene glycol results in extensive line-broadening of the resonances at -3 ppm and +1 ppm, but has no observable affect on the FAD resonances. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the activesite molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.

Animals↗
PubMed1984

Evidence for two copper atoms/subunit in dopamine beta-monooxygenase catalysis.

The stoichiometry of the copper requirement for the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine has been investigated by rapid chemical-quench techniques. This approach, which employs concentrated samples of enzyme, overcomes ambiguities of interpretation arising from levels of trace copper in excess of enzyme concentrations normally added to steady state kinetic assays. Low turnover numbers are observed when rapid quench kinetic studies are performed under conditions in which enzyme concentrations (2.5-7.1 microM) are in excess over trace copper levels (about 0.7 microM). The addition of exogenous Cu(II) results in full restoration of activity, which is maximal at a stoichiometry of 2 mol of copper/mol of enzyme subunit. From the dependence of catalytic activity on copper levels we conclude that both coppers are required for catalysis. No stimulation of activity was observed upon addition of the following metal ions: Ni(II), Co(II), Mn(II), Fe(III), and Zn(II). In addition, the magnitude of the tritium isotope effect for [2-3H]dopamine hydroxylation is invariant over a large range of enzyme activities accompanying changes in the ratio of copper to enzyme concentration. These results appear to rule out an effector role for the second mole of copper/subunit, implicating both copper atoms in active site redox chemistry.

Animals↗
PubMed1983

Kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

The kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase have been studied using the physiological substrates pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) at 25 degrees C and pH 8.0. Under steady-state conditions with different concentrations of PNP and O2, a series of parallel lines and competitive substrate inhibition with a KI of 50 microM are seen in double reciprocal plots. This is suggestive of a binary complex mechanism. Secondary plots yield a turnover number of 42 min-1 and Km values for both PNP (8.2 microM) and O2 (182 microM). A large deuterium isotope effect, VH/VD of 6.5, was observed with [4',4'-2H]PNP. In analogous studies using PMP, a turnover number of 6.2 min-1 and respective Km values for PMP and O2 of 3.6 and 85 microM were calculated. No significant substrate inhibition and a small deuterium isotope effect (VH/VD = 1.1) were observed with PMP. Anaerobic stopped flow data showed that the enzyme-bound flavin was reduced at a rate similar to catalytic turnover with PNP as a substrate, whereas with PMP, the rate of enzyme reduction is 500-fold faster than turnover. Stopped flow kinetic data also showed the reduced enzyme to react with O2 at rates at least 10(2)-10(3) faster than turnover. These results indicate that enzyme reduction is rate-limiting when the alcohol form (PNP) is the substrate, but in the case of the amine (PMP), the rate-limiting step in catalysis occurs subsequent to reduction. With PMP as substrate, release of product from the complex with reduced enzyme is 15-fold slower than turnover, and thus, it is suggested that oxygen reacts with the complex. The pH dependence of the deuterium isotope effect and the Km for PMP showed substantial change in the pH range between 6.0 and 7.5, whereas little or no pH dependence was observed for PNP. These data show that the kinetic mechanism of pyridoxamine (pyridoxine)-5'-phosphate oxidase can function via either a binary or ternary complex mechanism, depending upon the nature of the substrate.

Animals↗
PubMed1982

The role of the membrane in the regulation of activity of microsomal glucose-6-phosphatase.

The factors regulating glucose-6-phosphatase (EC 3.1.3.9) activity and substrate specificity in hepatic microsomes were studied by determining the rate-limiting reaction for the hydrolysis of glucose-6-P, and by examining the effect of detergent activation on phosphotransferase activity. Examination of the pre-steady state kinetics of glucose-6-phosphatase revealed that the steady state rate is determined by the rate of hydrolysis of the enzyme-P intermediate. Treatment of the enzyme with detergent does not alter the extent of the rapid release of glucose per mg of protein, but activates the steady state rate of catalytic turnover. Specificity of the enzyme was evaluated by comparing the effects of mannose and glucose as phosphate acceptors in the phosphotransferase reaction catalyzed by glucose-6-phosphatase. Untreated glucose-6-phosphatase discriminates against mannose as compared with glucose in that mannose and glucose bind to the enzyme-P intermediate of untreated enzyme, but mannose is not an acceptor of Pi. Mannose is an acceptor, however, after treatment of microsomes with detergent. These data cannot be explained in terms of the currently accepted "compartmentation" model for the regulation of glucose-6-phosphatase. The detergent-induced changes in kinetic properties appear to reflect alterations in the intrinsic characteristics of glucose-6-phosphatase, which could result from interaction with its membrane environment.

Animals↗
PubMed1981

8 alpha-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase.

8 alpha-(O-Tyrosyl)riboflavin has been synthesized by condensation of the copper complex of L-tyrosine with 8 alpha-bromotetraacetylriboflavin. The structure of this synthetic product was proven by absorption and 1H NMR spectroscopy and by chemical degradation, which yielded 1 mol of tyrosine per mol of flavin. The synthetic compound comigrated wtih the (aminoacyl)riboflavin isolated from the p-cresol methylhydroxylase of Pseudomonas putida, and both showed identical absorption and fluorescence spectral properties. 8 alpha-(O-Tyrosyl)riboflavin as well as the flavin-containing decapeptide from p-cresol methylhydroxylase undergoes reductive cleavage to form riboflavin and FAD, respectively, on anaerobic treatment with dithionite. In contrast, the native enzyme, on reduction with dithionite, yields a reduced flavin via a red (anionic) flavosemiquinone intermediate, which remains covalently bound to the protein even under denaturing conditions. 8 alpha-(O-Tyrosyl)riboflavin bound to apoflavodoxin is also not cleaved on reduction with dithionite, but, instead, a blue (neutral) semiquinone of tyrosylriboflavin is generated, which is resistant to further reduction with dithionite. Three p-cresol methylhydroxylases, isolated from different strains of Pseudomonas putida, differing in molecular weight and Km values for substrates, contain the same peptide at the flavin site. These data provide definitive proof for the existence of 8 alpha-(O-tyrosyl)riboflavin in nature.

Cresols↗