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

1980 ◽  
Vol 185 (3) ◽  
pp. 767-770 ◽  
Author(s):  
J P Malthouse ◽  
S Gutteridge ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
Computer Simulations ◽  
Active Centre ◽  
Geometrical Isomers ◽  
Paramagnetic Resonance ◽  
The Relationship ◽  
Electron Paramagnetic

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.

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

1979 ◽  
Vol 177 (1) ◽  
pp. 357-360 ◽  
Author(s):  
R C Bray ◽  
S Gutteridge ◽  
D A Stotter ◽  
S J Tanner
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
Low Ph ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Alternative Pathways ◽  
Ph Values ◽  
Covalent Intermediate ◽  
The Relationship ◽  
Electron Paramagnetic

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.

scholarly journals Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of aldehyde oxidase

1982 ◽  
Vol 203 (1) ◽  
pp. 263-267 ◽  
Author(s):  
R C Bray ◽  
G N George ◽  
S Gutteridge ◽  
L Norlander ◽  
J G Stell ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Aldehyde Oxidase ◽  
Resonance Spectroscopy ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic ◽  
Reduced Forms

Molybdenum(V) e.p.r. spectra from reduced forms of aldehyde oxidase were obtained and compared with those from xanthine oxidase. Inhibited and Desulpho Inhibited signals from aldehyde oxidase were fully characterized, and parameters were obtained with the help of computer simulations. These differ slightly but significantly from the corresponding parameters for the xanthine oxidase signals. Rapid type 1 and type 2 and Slow signals were obtained from aldehyde oxidase, but were not fully characterized. From the general similarities of the signals from the two enzymes, it is concluded that the ligands of molybdenum must be identical and that the overall co-ordination geometries must be closely similar in the enzymes. The striking differences in substrate specificity must relate primarily to structural differences in a part of the active centre concerned with substrate binding and not involving the catalytically important molybdenum site.

scholarly journals The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,6-diol) with the molybdenum centre of xanthine oxidase from electron-paramagnetic-resonance spectroscopy

1984 ◽  
Vol 218 (3) ◽  
pp. 961-968 ◽  
Author(s):  
T R Hawkes ◽  
G N George ◽  
R C Bray
Keyword(s):  
Nitrogen Atom ◽  
Xanthine Oxidase ◽  
Strong Coupling ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Kinetic Studies ◽  
Heterocyclic Ring ◽  
Resonance Spectroscopy ◽  
Active Centre ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Studies were carried out on the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine-4,5-diol) with xanthine oxidase, in extension of the work of Williams & Bray [Biochem. J. (1981) 195, 753-760]. By suitable regulation of the reaction conditions, up to 10% of the functional enzyme could be converted into the complex in the Mo(V) oxidation state. The e.p.r. spectrum of the complex was investigated in detail with the help of computer simulation and substitution with stable isotopes. Close structural analogy of the signal-giving species to that of the Very Rapid intermediate in enzyme turnover is shown by g-values (2.0279, 1.9593 and 1.9442) and by coupling to 33S in the cyanide-labile site of the enzyme [A(33S) 0.30, 3.10 and 0.70mT]. However, whereas in the Very Rapid signal there is strong coupling to 17O [Gutteridge & Bray, Biochem. J. (1980) 189, 615-623], instead, in the Alloxanthine signal there is strong coupling to a single nitrogen atom [A(14N) 0.35, 0.35, 0.32 mT]. This is presumed to originate from the 2-position of the heterocyclic ring system. From this work and from earlier kinetic studies it is concluded that alloxanthine, after being bound reversibly at the active centre, reacts slowly with it, in a specific manner, distinct from that in the normal catalytic reaction with substrates. This reaction involves elimination of an oxygen ligand of molybdenum and co-ordination, in this site, of alloxanthine via the N-2 nitrogen atom, to give a complex that is structurally but not chemically closely analogous to that of the Very Rapid species.

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

1981 ◽  
Vol 199 (3) ◽  
pp. 629-637 ◽  
Author(s):  
J P G Malthouse ◽  
G N George ◽  
D J Lowe ◽  
R C Bray
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Xanthine Oxidase ◽  
The Other ◽  
Reduced Form ◽  
Sulphur Atom ◽  
Paramagnetic Resonance ◽  
Different Types ◽  
Resonance Coupling ◽  
Electron Paramagnetic ◽  
Reduced Forms

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.

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