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A M Lambeir

Publications and source records attributed to A M Lambeir.

44 records · Page 3Linked to original sources
PubMed1985

Spectral properties of the higher oxidation states of prostaglandin H synthase.

Prostaglandin H (PGH) synthase reacts with organic hydroperoxides and fatty acid hydroperoxides on a millisecond time scale to generate an intermediate that is spectrally similar to compound I of horseradish peroxidase. Compound I of PGH synthase is converted to compound II within 170 ms. Compound II decays to resting enzyme in a few seconds. Thus, the peroxidase reaction of PGH synthase appears to involve a cycle of native enzyme, compound I, and compound II, typical of heme-containing peroxidases. The Soret absorption maximum of compound I appears to occur at 412 nm but a small amount of compound II may be present. Soret maxima occur at 420, 433, and 419 for compound II, the ferrous enzyme, and the oxyferrous enzyme (compound III), respectively. Rapid scan analysis of the reaction of PGH synthase with arachidonic acid reveals the absorbance of compound II but no evidence for ferrous or oxyferrous enzyme.

Alkenes↗
PubMed1985

Oxygen binding to dithionite-reduced chloroperoxidase.

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.

Chloride Peroxidase↗
PubMed1985

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase.

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.

Chemical Phenomena↗
PubMed1984

The reaction between NADPH and mercuric reductase from Pseudomonas aeruginosa.

The reaction between the FAD-containing enzyme, mercuric reductase, and its reducing substrate, NADPH, has been studied at 5 degrees C, pH 7.3, by rapid-scan and fixed-wavelength stopped-flow techniques with the aim to characterize reaction intermediates. With an excess of NADPH the spectral changes observed in rapid-scan experiments can be described by a simple kinetic model, A----B----C----D, where A, B, C, and D represent distinct spectral species. The first step is virtually complete within the dead time of the apparatus. It is associated with a 16% bleaching of flavin absorbance and the appearance of a very broad charge-transfer band (epsilon max = 1.9 mM-1 cm-1) centered near 600 nm. The second step (k = 43 s-1) involves a further small bleaching of flavin absorbance and intensification of the charge-transfer band (epsilon max = 3.3 mM-1 cm-1) which becomes centered near 580 nm. The third step (k = 8 s-1) involves an intensification and a blue shift of the main FAD absorption band. Concurrently, the charge-transfer band increases in intensity and becomes centered near 530 nm (epsilon 530 = 5.0 mM-1 cm-1). While the first two steps involve 1 mol of NADPH per mol of FAD, the third step requires a second equivalent of NADPH. A 2-electron-reduced enzyme (EH2) can be obtained by treatment of the oxidized enzyme (E) with dithioerythritol. Addition of 1 eq of NADP+ to dithioerythritol-generated EH2 gives rise to a spectral species similar to the kinetic intermediate C, while the addition of 1 eq of NADPH gives rise to a spectral species similar to D. It is proposed that B largely corresponds to an E-NADPH complex, C to an EH2-NADP+ complex, and D to an EH2-NADPH complex.

Hydrogen-Ion Concentration↗
PubMed1983

A steady state kinetic analysis of the reaction of chloroperoxidase with peracetic acid, chloride, and 2-chlorodimedone.

The chloroperoxidase-peracetic acid-chloride-2-chlorodimedone system is used as a model for the investigation of enzymatic halogenation reactions. Systematic variation of the concentrations of the three substrates under steady state conditions yields sets of kinetic parameters containing both kinetic and mechanistic information. Three distinct enzyme species are involved in the halogenation cycle: native enzyme, compound I, and a short-lived halogenating intermediate. Analysis of the kinetic data is complicated by the fact that chloride serves as a substrate in the second step and as an inhibitor of the first step of the overall halogenation reaction. The inhibitor binding site on the native enzyme must be protonated prior to the binding of chloride. Chloride appears to be a competitive inhibitor for both compound I formation and cyanide binding to chloroperoxidase. Only the latter reaction can be studied directly in stopped-flow experiments since compound I disappears rapidly by reacting with chloride present in solution. Rate constants are calculated for the individual steps of the reaction at four different pH values. The rate constant for compound I formation is independent of pH, but the actual rate is reduced at lower pH values due to pH dependence of the chloride inhibition. The rate constant for the oxidation of chloride by compound I decreases with increasing pH. The rate of the halogenation step appears to be independent of pH.

Acetates↗
PubMed1983

A kinetic and spectral study of the alkaline transitions of chloroperoxidase.

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.

Alkalies↗
PubMed1983

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase.

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.

Chloride Peroxidase↗