Properties of cupric ions in benzylamine oxidase from pig plasma as studied by magnetic-resonance and kinetic methods

Abstract

Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2.

Purification and Characterization of a Membrane-bound Deglycating Enzyme (1-Deoxyfructosyl Alkyl Amino Acid Oxidase, EC 1.5.3) from a Pseudomonas sp. Soil Strain

“…Similar to most copperdependent enzymes (such as cytochrome oxidase, amine oxidase/diamine oxidase, ascorbate oxidase, urate oxidase, galactose oxidase, phenolase, laccase, and dopamine B hydroxylase (21)(22)(23)(24)28)), this amadoriase is involved in an oxidoreduction type of reaction and is inhibited instantly by CN Ϫ and N 3 Ϫ . Cyanide binds directly to the copper (21)(22)(23)(24), and this enzyme also shows binding to 14 C-labeled cyanide (Fig. 3), suggesting the presence of a copper atom at its active site.…”

“…The enzyme was reacted with radiolabeled 14 C-cyanide, and protein-bound radioactivity was separated from free K 14 CN by Sephadex G-25. In similar experiments, radiolabeled cyanide has been ligated to Cu-containing amine oxidase (21), and cyanide has been shown to bind directly to copper (22)(23)(24). Nonspecific binding was checked with bovine serum albumin and found to be only ϳ9% of the specific binding to amadoriase (data not shown).…”

Enzymology of Quinoproteins

“…The latter is used as an artificial substrate in the assay (spectrophotometric determination of benzaldehyde formation). The molecular weight is about 170,000 and two subunits of closely similar composition were found (46). The enzyme contains two Cu2+ ions, which are intrinsically different and spatially separated (47).…”

Some Aspects of the Enzymic Inactivation of Sympathomimetic Amines

“…There is also considerable uncertainty concerning the dependence on copper in the enzyme mole cule. While some are known to contain this metal as an essential constituent [Barker et al, 1979], attempts to find it in rat BAT have failed , Doubts stem from the fact that some copper chelating agents are also carbonyl reagents. In addition, cyanide, which is also a chelator of copper [Crabbe et al, 1976] only inhibits some SSAO enzymes [Lyles and Callingham, 1975;Lewinsohn, 1984], Attempts to determine the substrate selectivities of the SSAOs have not produced a clear picture, except to show that many of these enzymes prefer the physiologically in significant amine, benzylamine.…”