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R C Bray

Publications and source records attributed to R C Bray.

At least 127 records · Page 7Linked to original sources
PubMed1982

The extents of formation of cobalt(II)-radical intermediates in the reactions with different substrates catalysed by the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase.

1. The reactions of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, with the 'good' and 'relatively poor' substrates 2-aminoethanol and (S)-2-aminopropanol respectively, under conditions of saturation with substrate were investigated by rapid freezing in conjunction with electron paramagnetic resonance (e.p.r.) spectroscopy and by stopped-flow spectrophotometry. 2. In disagreement with earlier reports [Babior et al. (1972) J. Biol. Chem. 247, 4389-4392], it was found that the reaction of 2-aminoethanol gave an e.p.r. signal observed in rapid freezing experiments characteristic of a coupled Co(II)-free radical system. This signal was similar to, though not identical with, that obtained with (S)-2-aminopropanol. The steady-state level of the signal with 2-aminoethanol as substrate was 0.56 of that attained with (S)-2-aminopropanol. 3. The results of these e.p.r. experiments were shown to be consistent with stopped-flow data obtained under closely similar reaction conditions, the latter indicating a corresponding ratio of 0.64. The results also are consistent with those of a rapid wavelength scanning, stopped-flow spectrophotometric study [Hollaway et al. (1978) Eur. J. Biochem. 82, 143-154].

Ammonia-Lyases↗
PubMed1981

Coupling of [33S]sulphur to molybdenum(V) in different reduced forms of xanthine oxidase.

Different reduced forms of xanthine oxidase, labelled specifically in the cyanide-labile site with 33S, were prepared and examined by electron paramagnetic resonance. Coupling of this isotope to molybdenum(V) was quantified with the help of computer simulations and found to differ markedly from one reduced form to another. The xanthine Very Rapid signal shows strong, highly anisotropic, coupling with A(33S)av. 1.27 mT. For this signal, axes of the g- and A(33S)-tensors are rotated relative to one another. One axis of the A-tensor is in the plane of gxx ang gyy, but rotated by 40 degrees relative to the gxx axis, whereas the direction of weakest coupling to sulphur deviates by 10 degrees from the gzz axis. In contrast with this signal, only rather weaker coupling was observed in different types of Rapid signal [A(33S)av. 0.3--0.4 mT], and in the Inhibited signal coupling was weaker still [A(33S)av. 0.1--0.2 mT]. Clearly, there must be substantial differences in the structures of the molybdenum centre in the different signal-giving species, with the sulphur atom perhaps in an equatorial type of ligand position in the Very Rapid species but in a more axial one in the other species. Structures are discussed in relation to the mechanism of action of the enzyme and the nature of the proton-accepting group that participates in turnover.

Chemical Phenomena↗
PubMed1981

Molybdenum(V) e.p.r. signals obtained from xanthine oxidase on reduction with aldehyde substrates and with 2-amino-4-hydroxy-6-formylpteridine.

2-Amino-4-hydroxy-6-formylpteridine, a known 'slow' substrate and inhibitor of xanthine oxidase, is unusual in that it gives rise under suitable conditions to all types of molybdenum(V) e.p.r. signals obtainable from the enzyme, namely Very Rapid, Rapid, Inhibited and Slow. The Very Rapid signal appears in a slightly modified form. The Inhibited signal, originally thought to be unique to reaction of methanol or of formaldehyde with xanthine oxidase, is now shown to be obtainable with several other aldehydes. These include, in addition to 2-amino-4-hydroxy-6-formylpteridine, acetaldehyde and glycoaldehyde. Parameters of the signals, obtained with the help of computer simulations, are presented. The appearance of Very Rapid and of Inhibited signals with these additional substrates may be of importance in elucidating the structure of the enzyme active centre. In agreement with previous work, the Very Rapid signal is attributed to an obligatory intermediate in turnover. On the other hand, the Inhibited signal is attributed to a side reaction, presumably inhibitory in nature, occurring during the catalytic process.

Aldehydes↗
PubMed1981

Kinetic and e.p.r. studies on the inhibition of xanthine oxidase by alloxanthine (1 H-pyrazolo [3, 4-d] pyrimidine-4,6-diol).

The inhibition by alloxanthine of oxidation of xanthine by xanthine oxidase is characterized by a prolonged transient phase. Kinetic data accord with a mechanism that involves rapid formation of a reduced enzyme-alloxanthine complex that subsequently undergoes a relatively slow-reversible reaction. In this scheme the slowly formed complex cannot be fully reoxidized by oxygen. From the Ki value for the dissociation of alloxanthine from the rapidly formed complex (1.15 microM) and values of 0.37 min-1 and 0.011 min-1 for the forward and reverse rate constants of the slow reaction, an overall inhibition constant for alloxanthine of 35 nM was calculated. A molybdenum (V) e.p.r. signal from the slowly formed reduced enzyme-alloxanthine complex is described. The rate of appearance of this new signal is consistent with this assignment. The signal (the "Alloxanthine signal") was simulated with g1 2,0269, g2 1,9593, g3 11.9444 and shows indications of hyperfine coupling to nitrogen. Similarities between it and the Very Rapid signal are discussed. Close structural analogies between the catalytic intermediate represented by the Very Rapid signal and the inhibitor complex represented by the Alloxanthine signal are suggested.

Dithionite↗
PubMed1980

X-ray absorption spectroscopy of xanthine oxidase. The molybdenum centres of the functional and the desulpho forms.

X-ray absorption spectra have been recorded for the molybdenum K-edge region of xanthine oxidase. Both the absorption edge and the extended fine structure (e.x.a.f.s.) regions were investigated. Spectra were obtained for samples of the desulpho enzyme as well as for mixtures of this with the active enzyme. The spectrum of the pure active form was then obtained by difference. The desulpho enzyme shows a pronounced step in the absorption edge, of a type previously associated terminal oxygen ligands. In the active enzyme this step has decreased markedly. Satisfactory simulations of the e.x.a.f.s. spectrum of the desulpho enzyme could be obtained by assuming the molybdenum to be bonded to two terminal oxygen atoms (Mo = O about .175 nm), two sulphur atoms (presumably from cysteine residues, Mo-S about .0250 nm) and one sulphur atom (presumably from a methionine residue, Mo-S about 0.290 nm). E.x.a.f.s. of the active enzyme differed appreciably from this. In keeping with earlier proposals [Gutteridge, Tanner & Bray (1978) Biochem. J. 175, 887-897], the spectrum of the active enzyme could be simulated if a sulphur atom at about 0.225 nm (i.e. presumably a terminal sulphur atom) replaced one of the terminal oxygen atoms of the desulpho from, with small changes in the other bond distances. Validity of the interpretative procedures, which involved phase shift and amplitude calculations ab initio, was demonstrated by using low molecular weight compounds of known structure.

Chemical Phenomena↗
PubMed1980

The nature of the sulphur atom liberated from xanthine oxidase by cyanide. Evidence from e.p.r. spectroscopy after 35S substitution.

Active xanthine oxidase was labelled specifically with 33S in the cyanide-labile site of the molybdenum centre. The Very Rapid molybdenum (V) e.p.r. signal, generated from this, shows strong coupling of 33S to molybdenum, providing unambiguous evidence that, at least in the signal-giving species, this sulphur atom is a ligand of molybdenum. The structure of the signal-giving species is discussed.

Chemical Phenomena↗
PubMed1980

The nature of the phosphate inhibitor complex of sulphite oxidase from electron-paramagnetic-resonance studies using oxygen-17.

Studies of the effect of substitution with 17O on the e.p.r. spectra at 9 and 35 GHz of Mo(V) in the phosphate complex of sulphite oxidase are reported. Substitution of 17O-enriched water for normal water, for samples of the enzymes reduced by sulphite in the presence of normal phosphate, produced no detectable effect on the e.p.r. signal. If phosphate substituted with 17O was used, coupling due to 17O, producing large anisotropic splittings in the spectrum, was clearly detectable. It is concluded that phosphate is co-ordinated directly to molybdenum in the active site of the enzyme, in an equatorial type of ligand position. An oxygen ligand must be displaced from the molybdenum in the process of binding the phosphate. Implications concerning the mechanism of the enzyme reactions are discussed.

Binding Sites↗
PubMed1980

Oxygen-17 splitting of the very rapid molybdenum(V) e.p.r. signal from xanthine oxidase. Rate of exchange with water of the coupled oxygen atom.

Studies have been carried out of effects of 17O substitution on a Mo(V) e.p.r. signal from xanthine oxidase, known as Very Rapid. This transient signal is believed to represent an intermediate in enzymic turnover. When Very Rapid was developed from enzyme equilibrate with 17O-enriched water, strong coupling of Mo(V) to a single oxygen atom was observed, with A(17O)1,2,3 1.34, 1.40, 1.36 mT. The isotropic character of the splittings is interpreted as favouring a structure of the type Mo--O--C. The rate of exchange with water of the oxygen atom detected in the signal was studied. In oxidized enzyme, which contains a terminal oxygen ligand, the exchange rate constant was 2--4 h-1 (pH 5.9--7.8 and about 20 degrees C). However, if the exchange was allowed to take place whilst the enzyme was turning over a substrate, then the process occurred within a few seconds. The present and previous results are interpreted as favouring an enzymic mechanism in which a terminal oxygen ligand reacts, as a nucleophile, with a substrate carbonium ion. To complete the reaction, product liberation, by hydrolysis of the enzyme-bound species, occurs in such a way as to cleave the Mo--O bond, thus explaining the fast oxygen exchange in the presence of the substrate.

Aldehydes↗
PubMed1980

Rapid type 2 molybdenum(V) electron-paramagnetic resonance signals from xanthine oxidase and the structure of the active centre of the enzyme.

Rapid type 2 molybdenum(V) e.p.r. signals from reduced functional xanthine oxidase have been further investigated. These signals, which show strong coupling of two protons to molybdenum, have been obtained under a variety of new conditions: specifically either at pH 8.2 in the presence of borate ions, or at pH 10.1--10.7 with or without various other additions. Parameters of the signals were obtained with the help of computer simulations. In at least some of these signals, the coupled protons must be located on the enzyme rather than on bound species. The relationship between type 1 and type 2 Rapid signals is discussed. They may represent geometrical isomers, or alternatively, hydroxyl uptake as a ligand of molybdenum may be involved in formation of type 2 species.

Binding Sites↗
PubMed1979

Purification and properties of nitrogenase in ethylene glycol at sub-zero temperatures.

Both the protein components Kp1 and Kp2 of nitrogenase from Klebsiella pneumoniae were found to be stable in aq. 50% (v/v) ethylene glycol at +30 degrees C or below. At -20 degrees C in this medium their sensitivities to O2 were diminished somewhat. Though purification could be carried out at -20 degrees C, the product had the same specific activity and was obtained in the same yield as when the purification was carried out by standard procedures. This suggests that such procedures yield enzyme undamaged in the course of the purification by O2, thermal denaturation or proteolytic digestion.

Chemical Phenomena↗
PubMed1979

The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals.

On the basis of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897] and of other data in the literature, a mechanism for the reaction of xanthine oxidase with reducing substrates is proposed. In the Michaelis complex, xanthine is bound to molybdenum via the N-9 nitrogen atom. Coupled transfer of two electrons to molybdenum and the C-8 proton to the enzyme yields (Enzyme)-Mo-SH. Concerted with this process, reaction of the xanthine residue with a nucleophile in the active centre yields a covalent intermediate that breaks down to give the product by alternative pathways at high and at low pH values.

Chemical Phenomena↗
PubMed1979

EXAFS studies of the molybdenum center of xanthine oxidase.

EXAFS spectra associated with the K-absorption edge of molybdenum in the desulpho and functional forms of xanthine oxidase and some potential synthetic analogues have been obtained. These data indicate that the immediate environment of the molybdenum is different in the two forms of the enzyme and that desulpho xanthine oxidase resembles that in [MoO2(S2CNEt2)2] and [MoO2(ethylcysteine)2]. The cyanolysable sulphur atom of functional xanthine oxidase is suggested to be tightly bound to the molybdenum at a distance of less than or equal to 2.3 A.

Binding Sites↗
PubMed1978

The molybdenum centre of native xanthine oxidase. Evidence for proton transfer from substrates to the centre and for existence of an anion-binding site.

The observation by Bray & Knowles [Proc. R. Soc. London Ser. A (1968) 302, 351--353] of direct transfer, during the catalytic reaction, of hydrogen atoms from substrate molecules to the enzyme xanthine oxidase was reinvestigated. The experimental phenomenon and its basic interpretation were confirmed and extended. In the reduced functional enzyme, molybdenum(V) interacts with two enzyme-bound protons, which are exchangeable with solvent protons. One of these is coupled to the metal with AHav. 1.4mT and the other with AHav. 0.3mT. The molecule also contains a site for the binding of anions, presumably as ligands of molybdenum. This is shown by effects of nitrate ions on the e.p.r. spectra. The spectra of the nitrate and 1-methylxanthine complexes of the reduced enzyme are very similar to one another, and are designated Rapid type-1 spectra. It is concluded that, in the Michaelis complex, the substrate molecule occupies the anion site, probably being bound to molybdenum via the nitrogen in its 9-position. During the turnover process, hydrogen from the substrate C-8 position, after transfer to the enzyme, appears as the proton more strongly coupled to molybdenum. This proton then exchanges with solvent deuterium with a rate constant of 27s-1, at pH 8.2 and 12 degrees C. It has been confirmed that substrate molecules occupying the anion site do not interfere with observation of the transfer and exchange processes.

Anions↗
PubMed1978

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.

Chemical Phenomena↗
PubMed1978

Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group.

The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.

Anions↗
PubMed1978

Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12.

Nitrate reductase was purified from anaerobically grown Escherichia coli K12 by a method based on the Triton X-100 extraction procedure of Clegg[(1976) Biochem. J.153, 533-541], but hydrophobic interaction chromatography was used in the final stage. E.p.r. spectra obtained from the enzyme under a variety of conditions are well resolved and were interpreted with the help of the computer-simulation procedures of Lowe [(1978) Biochem. J.171, 649-651]. Parameters for five molybdenum(V) species from the enzyme are given. The low-pH species (g(av.) 1.9827) is in pH-dependent equilibrium with the high-pH species (g(av.) 1.9762), the pK for interconversion of the species being 8.26. Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified molybdenum(V) e.p.r. spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton. The fifth molybdenum(V) species, sometimes detected in small amounts, appears not to be due to functional nitrate reductase. After full reduction of the enzyme with dithionite, addition of nitrate caused reoxidation of molybdenum to the quinquivalent state, in a time less than the enzyme turnover. Activity of the enzyme in the pH range 6-10 is controlled by a pK of 8.2. It is suggested that the low-pH signal-giving species is the form of the enzyme involved in the catalytic cycle. Iron-sulphur and other e.p.r. signals from the enzyme are briefly described and the enzymic reaction mechanism is discussed.

Chemical Phenomena↗
PubMed1978

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

Animals↗