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Reactions of bovine serum amine oxidase with NN-diethyldithiocarbamate. Selective removal of one copper ion.

ABSTRACT: NN-Diethyldithiocarbamate (DDC) was able to bind, at 1.0 mM concentration, only about 50% the Cu(II) ions of bovine plasma amine oxidase. Under reducing conditions, this Cu(II) was removed with inactivation of the enzyme. Up to 90% activity could be recovered by treatment with excess Cu(II). The organic cofactor, sensitive to carbonyl reagents, was reduced in the half-Cu-depleted protein and no longer bound phenylhydrazine. The fully reacted protein, in the presence of 10 mM-DDC, lost 50% Cu(II) upon storage at -20 degrees C, but in this case the residual Cu(II) was in the DDC-bound form and the cofactor was in the oxidized state, as it could still bind phenylhydrazine. In the presence of DDC, the rate of reaction with phenylhydrazine was always low, even at 50% DDC saturation, and all derivatives showed identical modifications of the optical and e.p.r. spectra with respect to the phenylhydrazone of the native protein. It is concluded that the two Cu(II) ions are not equivalent, that removal of a single Cu(II) is sufficient to inhibit the re-oxidation of the organic cofactor, and that both Cu(II) ions are in some way involved in the reaction with phenylhydrazine. After reaction with DDC, the optical and e.p.r. spectra of 63Cu(II)-amine oxidase and of 63Cu(II)-carbonic anhydrase [Morpurgo, Desideri, Rigo, Viglino & Rotilio (1983) Biochim. Biophys. Acta 746, 168-175] are very similar and show distorted equatorial co-ordination to Cu(II) of two sulphur atoms and two magnetically equivalent nitrogen atoms.

SUBMITTER: Morpurgo L

PROVIDER: S-EPMC1148629 | biostudies-other | 1987 Dec

REPOSITORIES: biostudies-other

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Reactions of the oxidized organic cofactor in copper-depleted bovine serum amine oxidase.

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Spectroscopic studies of the reaction between bovine serum amine oxidase (copper-containing) and some hydrazides and hydrazines.

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Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.

Project description:Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5-15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3-5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.

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