Biomedical subjects

E Lloyd Raven

Publications and source records attributed to E Lloyd Raven.

4 recordsLinked to original sources

PubMed2001

Engineering the active site of ascorbate peroxidase.

Understanding the catalytic versatility of haem enzymes, and in particular the relationships that exist between different classes of haem-containing proteins and the mechanisms by which the apo-protein structure controls chemical reactivity, presents a major experimental and theoretical challenge. These issues are discussed in the general context of peroxidase and cytochrome P450 chemistry, and specific issues relating to the catalytic chemistry of ascorbate peroxidase are highlighted.

Ascorbate Peroxidases↗

PubMed2001

Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide.

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.

Ascorbate Peroxidases↗

PubMed2000

Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide.

The activity of ascorbate peroxidase (APX) has been studied with H(2)O(2) and various reducing substrates. The activity decreased in the order pyrogallol>ascorbate>guaiacol>2, 2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H(2)O(2) as the sole substrate was studied. The number of H(2)O(2) molecules required for maximal inactivation of the enzyme was determined as approx. 2.5. Enzymic activity of approx. 20% of the original remained at the end of the inactivation process (i.e. approx. 20% resistance) when ascorbate or ABTS was used as the substrate in activity assays. With pyrogallol or guaiacol no resistance was seen. Inactivation by H(2)O(2) followed over time with ascorbate or pyrogallol assays exhibited single-exponential decreases in enzymic activity. Hyperbolic saturation kinetics were observed in both assay systems; a similar dissociation constant (0.8 microM) for H(2)O(2) was obtained in each case. However, the maximum rate constant (lambda(max)) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H(2)O(2) partly or completely protected the enzyme from inactivation, depending on how many molar equivalents of reducing substrate were added. An oxygen electrode system has been used to confirm that APX does not exhibit a catalase-like oxygen-releasing reaction. A kinetic model was developed to interpret the experimental results; both the results and the model are compared and contrasted with previously obtained results for horseradish peroxidase C. The kinetic model has led us to the conclusion that the inactivation of APX by H(2)O(2) represents an unusual situation in which no enzyme turnover occurs but there is a partition of the enzyme between two forms, one inactive and the other with activity towards reducing substrates such as ascorbate and ABTS only. The partition ratio is less than 1.

Ascorbate Peroxidases↗

PubMed2000

Catalytic oxidation of p-cresol by ascorbate peroxidase.

Transient and steady state kinetics, together with a range of chromatographic and spectroscopic techniques, have been used to establish the mechanism and the products of the H(2)O(2)-dependent oxidation of p-cresol by ascorbate peroxidase (APX). HPLC, GC-MS, and NMR analyses are consistent with the formation of 2, 2'-dihydroxy-5,5'-dimethylbiphenyl (II) and 4alpha,9beta-dihydro-8, 9beta-dimethyl-3(4H)-dibenzofuranone (Pummerer's ketone, III) as the major products of the reaction. In the presence of cumene hydroperoxide, two additional products were observed which, from GC and MS analyses, were shown to be 1,1-dimethylbenzylalcohol (IV) and bis-(1-methyl-1-phenyl-ethyl)-peroxide (V). The product ratio II:III was dependent on enzyme concentration: at low concentrations Pummerer's ketone (III) predominates and at high concentrations formation of the biphenyl compound (II) is favored. Steady-state data showed a sigmoidal dependence on [p-cresol] that was consistent with the presence of 2.01 +/- 0.15 binding sites for the substrate (25.0 degrees C, sodium phosphate, pH 7.0, mu = 2.2 mM) and independent of ionic strength in the range 2.2-500 mM. Single turnover kinetic experiments (pH 7.0, 5.0 degrees C, mu = 0.10 M) yielded second-order rate constants for Compound I reduction by p-cresol, k(2), of 5.42 +/- 0.10 x 10(5) M(-1) s(-1), respectively. Rate-limiting reduction of Compound II by p-cresol, k(3), showed saturation kinetics, giving values for K(d) = 1.54 +/- 0.12 x 10(-3) M and k(3) = 18.5 +/- 0.7 s(-1). The results are discussed in the more general context of APX-catalyzed aromatic oxidations.

Ascorbate Peroxidases↗