Properties of cobalt‐substituted bovine serum amine oxidase

Abstract

Half-copper-depleted and fully copper-depleted amine oxidase from bovine serum were reconstituted with either copper or cobalt. All samples were studied by high-sensitivity scanning calorimetry, by enzyme activity analysis, and by reactivity with phenylhydrazine. The calorimetric profile of the protein was strongly modified by the removal of a single Cu ion approximately to the same extent as by complete copper removal, in agreement with the loss of over 80% enzymic activity. The thermograms of metal-reconstituted species showed a marked similarity with that of the native enzyme, irrespective of whether copper or cobalt was present. Reactivity with phenylhydrazine and enzymic activity measurements showed that in cobalt-substituted amine oxidase the organic cofactor was reactive and the enzyme was catalytically competent, although kinetically less efficient. These observations agree both with previous findings on the protein half-site reactivity and with previous suggestions for a copper conformational role in bovine serum amine oxidase, namely of maintaining a functional conformation at the active site.

Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains

“…The DSC curve for LSAO shows a small and broad transition below 55 °C, the enthalpy of which is consistent with a conformational change in the native enzyme, whereas for BSAO there is negligible variation in the same range of temperature [37,39], which is in agreement with the specific activity variation studies previously reported [40]. The deconvolution of the excess molar heat capacity reveals in LSAO and BSAO three subpeaks (transitions I-III) and five subpeaks respectively [37,36]. In LSAO the subpeaks (energetic domains, transitions I,II and III) [41] may belong to the three hypothesized structural domains for each subunit of LSAO (D3 , D2 and D4 respectively) [5].…”

“…Since Co(II)-containing BSAO, like Co(II)-substituted LSAO, cannot form a stable semiquinone radical, it can be excluded that the formation of the Cu(I)-semiquinone is an essential step of the BSAO catalytic pathway [13]. That the metal in BSAO is not involved in amine oxidation process, but shows a structural role, was also confirmed by the similar behaviour of Cu(II)-and Co(II)-BSAO derivatives in differential scanning calorimetry studies [13,36].…”

Modeling Novel Quinocofactors: An Overview

“…Reconstitution of copper-depleted BSAO with Co(II) was found to restore some amine oxidase activity (57,65,66). Since the Co(II)-derivative cannot form a semiquinone radical intermediate, the finding that Co(II)-BSAO is catalytically competent suggests that the Cu(I)-semiquinone is not an obligatory intermediate of the catalytic pathway.…”

“…Since the Co(II)-derivative cannot form a semiquinone radical intermediate, the finding that Co(II)-BSAO is catalytically competent suggests that the Cu(I)-semiquinone is not an obligatory intermediate of the catalytic pathway. On the basis of these findings, the metal ion in BSAO was suggested to have a structural function, i.e., to maintain the active site in a proper conformation, rather than being directly involved in amine oxidation (57,65). More recently, Sue and Klinman conducted a very detailed study on the oxidative-half reaction of BSAO and proposed a passive role of copper in the reoxidation of reduced cofactor by molecular oxygen (67).…”

Electroactive Centers inEuphorbiaLatex and Lentil Seedling Amine Oxidases

“…Copper-depleted enzymes were prepared as previously described. 19,20) Briefly, dithionite was added to solutions of the enzymes under anaerobic conditions, followed by dialysis against a solution of 1.0 mM sodium cyanide in 100 mM potassium phosphate buffer, pH 7.0.…”