On the origin of the cyanolysable sulphur in molybdenum iron/sulphur flavin hydroxylases

Coughlan, Michael P.

doi:10.1016/0014-5793(77)80914-9

Cited by 21 publications

(6 citation statements)

References 20 publications

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“…Bray & Swann (1972) suggested that the sulphur might be a ligand of molybdenum. More specifically, Coughlan (1977) proposed cysteine sulphur liganded to molybdenum. However, evidence that this could give rise to thiocyanate, on treatment with cyanide, seems slight.…”

Section: (Received 12 May 1978)mentioning

confidence: 99%

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

Gutteridge¹,

Tanner²,

Bray³

1978

Biochemical Journal

131

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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.

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Section: (Received 12 May 1978)mentioning

confidence: 99%

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

Gutteridge¹,

Tanner²,

Bray³

1978

Biochemical Journal

131

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“…The thiol reagent used at 2.5M concentration inactivates also xanthine oxidase from milk (Harris & Hellerman, 1956). Possibly this reagent reacts with the thiol group of cysteine in the proximity of the molybdenum centre involved in hydroxylation of purine substrate (Coughlan, 1977;Coughlan etal., 1980). On the other hand, 4-chloromercuribenzoate at concentrations up to 5 ,M increased the total activity of the enzyme preparation.…”

Section: Resultsmentioning

confidence: 99%

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity

Kamiński¹,

Jeżewska²

1982

Biochemical Journal

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The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.

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“…Edmondson et al [31] have proposed that the S-binding covalently to the carbon is part of a persulphide group proposed by Massey and Edmondson [32]. We prefer the proposal of Coughlan [33] that the -S -is part of a cysteinyl residue which is bound to the molybdenum but when the substrate reacts at the active site the bond between the cysteinyl sulphur and the molybdenum is broken. This explanation is preferred since according to the model presented here the sulphur and the molybdenum are 0.35 nm apart; thus if there is any movement of these atoms they could easily come within the bonding distance of 0.23 nm [34].…”

Section: The Geometry O J the Active Sitementioning

confidence: 97%

A Mutation in the Xanthine Dehydrogenase (Purine Hydroxylase I) of Aspergillus nidulans Resulting in Altered Specificity. Implications for the Geometry of the Active Site

Scazzocchio

Sealy-Lewis

1978

Eur J Biochem

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A point mutation in the structural gene for purine hydroxylase I (xanthine dehydrogenase) of Aspergillus nidulans results in several dramatic pleiotropic effects. The mutant enzyme oxidises 2-hydroxypurine at position 6 rather than 8, shows a 70-fold reduction in the I/ for hypoxanthine, and loses the ability to accept xanthine as a substrate. Allopurinol, a powerful pseudoirreversible inhibitor of the wild type enzyme, behaves as a good substrate of the mutant enzyme. We propose that the substrate and inhibitor specificities of the enzymes depend on the relative position of an orientating site and a catalytic site. All the properties of the mutant enzyme can be explained by assuming that the mutation results in a change of the relative positions of the catalytic and orientating sites.We have assumed that the catalytic site comprises a Mo(V1) atom and an -S-group as proposed by Coughlan [FEBS Lett. 81, 1-9 (1977)l and the orientating site is a lysyl residue. While these assumptions are not strictly necessary for the construction of an abstract geometric model, they are consistent with other data bearing on the structure of the active site of the molybdenum-containing hydroxylases.

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On the origin of the cyanolysable sulphur in molybdenum iron/sulphur flavin hydroxylases

Cited by 21 publications

References 20 publications

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

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

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity

A Mutation in the Xanthine Dehydrogenase (Purine Hydroxylase I) of Aspergillus nidulans Resulting in Altered Specificity. Implications for the Geometry of the Active Site

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