Biomedical subjects

E L Raven

Publications and source records attributed to E L Raven.

4 recordsLinked to original sources

PubMed2001

Engineering the active site of ascorbate peroxidase.

The oxidation of a number of thioethers, namely methyl phenyl sulphide (1), ethyl phenyl sulphide (2), isopropyl phenyl sulphide (3), n-propyl phenyl sulphide (4), p-chlorophenyl methyl sulphide (5), p-nitrophenyl methyl sulphide (6) and methyl naphthalene sulphide (7), by recombinant pea cytosolic ascorbate peroxidase (rAPX) and a site-directed variant of rAPX in which the distal tryptophan 41 residue has been replaced with an alanine (W41A) has been examined. The electronic spectrum (pH 7.0, mu = 0.10 M, 25.0 degrees C) for the ferric derivative of W41A (lambda(max)/nm = 411, 534, 560, 632) is indicative of an increased quantity of 6-coordinate, high-spin and/or 6-coordinate, low-spin haem compared to rAPX. Steady state oxidation of sulphides 1-4 and 7, gave values for kcat that are approximately 10-fold and 100-fold, respectively, higher for W41A than for rAPX. For rAPX, essentially racemic mixtures of R- and S-sulphoxides were obtained for all sulphides. With the exception of sulphide 7, the W41A variant shows substantial enhancements in enantioselectivity, with R : S ratios varying between R : S = 63 : 37 (sulphides 1 and 4) and R : S = 85 : 15 (sulphide 6). Incubation of sulphide 2 with rAPX or W41A and [(18)O] H(2)O(2) shows 95% (rAPX) and 96% (W41A) transfer of labelled oxygen to the substrate. Structure-based modelling techniques have provided a fully quantitative rationalization of all the experimentally determined R : S ratios and have indicated that reorientation of the sidechain of Arg38, such that access to the haem is much less restricted, is influential in controlling the stereoselectivity for both rAPX and W41A.

Amino Acid Substitution↗

PubMed2000

Peroxidase-catalyzed oxidation of ascorbate. Structural, spectroscopic and mechanistic correlations in ascorbate peroxidase.

Ascorbate-dependent peroxidase activity was first reported in 1979 (Groden and Beck, 1979; Kelly and Latzko, 1979) and ascorbate peroxidase (APX) is, therefore, a relative newcomer to the peroxidase field--horseradish (HRP) and cytochrome c (CcP) peroxidases were, for example, first identified in 1903 (Bach and Chodat, 1903) and 1940 (Altschul et al., 1940) respectively. The APX area was reviewed by Dalton in 1991 (Dalton, 1991): at that time, there was very little detailed kinetic, spectroscopic or functional information available and no structural information had been published. Since 1991, there have been some major advances in the field, most notably with the publication, in 1995, of the first crystal structure for an APX enzyme (Patterson and Poulos, 1995). This information, together with the availability of new recombinant expression systems (Yoshimura et al., 1998; Caldwell et al., 1998; Dalton et al., 1996; Patterson and Poulos, 1994), served as a catalyst for the publication of new functional and spectroscopic data and has meant these data could be sensibly rationalized at the molecular level. The aim of this review is to summarize the more recent advances in the APX area and, as far as possible, to draw comparisons with other, more well-characterized peroxidases. The review will concentrate on the ways in which structural, spectroscopic and mechanistic information have been used in a complementary way to provide a more detailed picture of APX catalysis. The more biological and physiological aspects of APX enzymes have been previously covered in a comprehensive manner (Dalton, 1991) and will not, therefore, be dealt with in detail here.

Ascorbate Peroxidases↗

PubMed2000

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.

Base Sequence↗

PubMed1998

Class I heme peroxidases: characterization of soybean ascorbate peroxidase.

An efficient expression system [D. A. Dalton et al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for detailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry. Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 K. Low-temperature EPR (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction potential for the Fe(III)/Fe(II) redox couple, determined by spectroelectrochemistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu = 0.10 M). Circular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degrees C. These results represent the first detailed spectroscopic and electrochemical analysis of soybean ascorbate peroxidase and are discussed in the broader context of other class I peroxidases.

Amino Acid Sequence↗