Preparation and Characterization of Cobalt(II)-Substituted Bovine Serum Amine Oxidase1

Suzuki, Shinnichiro; Sakurai, Takeshi; Nakahara, Akitsugu; Oda, Osamu; Manabe, Takashi; Okuyama, Tsuneo

doi:10.1093/oxfordjournals.jbchem.a133551

Cited by 14 publications

(12 citation statements)

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“…The CD spectrum of Co# + -BSAO, prepared by dialysis against excess Co# + of the protein depleted of Cu# + by cyanide and dithionite (Figure 3, spectrum a), is similar to the previously reported one, prepared from the fully reduced apo protein [29]. It shows a band at 540 nm, which, like the band at 640 nm of the native protein (Figure 3, curve b), was assigned as an Me# + d-d electronic transition [20,29]. The band at 440 nm, occurring in both spectra, was assigned as a TPQ transition [29].…”

Section: Spectroscopic Properties Of Co 2 + -Bsaosupporting

confidence: 80%

“…It shows a band at 540 nm, which, like the band at 640 nm of the native protein (Figure 3, curve b), was assigned as an Me# + d-d electronic transition [20,29]. The band at 440 nm, occurring in both spectra, was assigned as a TPQ transition [29]. The lack in spectrum (a) of appreciable intensity in the region where Cu# + absorbs confirms that the Co# + derivative does not contain more than 10 % Cu# + (Table 1).…”

Section: Spectroscopic Properties Of Co 2 + -Bsaomentioning

confidence: 87%

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Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Agostinelli¹,

Matteis²,

Mondovı̀³

et al. 1998

Biochemical Journal

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Two different Cu2+-depleted derivatives of bovine serum amine oxidase (BSAO) have recently been prepared, which contain about 0.5 mol/dimer of phenylhydrazine-reactive topa quinone (TPQ) cofactor and, depending on the reagents used, about 0.2 or 0.7 residual Cu2+/dimer [Agostinelli, De Matteis, Sinibaldi, Mondovi and Morpurgo (1997) Biochem. J. 324, 497-501]. The benzylamine oxidase activity of both derivatives was <5% and increased up to approximately 20% on incorporation of Co2+, irrespective of the residual Cu2+ content, which was unaffected by the treatment according to atomic absorption and ESR spectroscopy. The residual Cu2+ ions appeared to be distributed one per dimer and to be bound to inactive subunits, whereas Co2+ was bound to active subunits. The change in the active site had an appreciable influence on the kinetic behaviour. With several amines, the kinetic parameters, Km and kc, measured for Co2+-BSAO were different from those for native BSAO. This excludes the possibility that the catalytic activity was due to residual Cu2+. Furthermore, Co2+ restored to nearly native level the intensity of the TPQ 480 nm band and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu2+-depleted samples. The CD spectrum, measured for the derivative with low Cu2+ content, was compatible with Co2+ binding to the copper site. The amine oxidase activity of the Co2+ derivative, which cannot form a semiquinone radical as an intermediate of the catalytic reaction, strongly suggests that the Cu+-semiquinone is not an obligatory intermediate of BSAO catalytic pathway.

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Section: Spectroscopic Properties Of Co 2 + -Bsaosupporting

confidence: 80%

Section: Spectroscopic Properties Of Co 2 + -Bsaomentioning

confidence: 87%

Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Agostinelli¹,

Matteis²,

Mondovı̀³

et al. 1998

Biochemical Journal

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“…For Cu-ECAO and Ni-ECAO there was no effect, while for Co-ECAO we observed a modest increase in k cat from 0.27 to 0.43 s −1 ; however, this is significantly different from the value of 9.8 s −1 for Cu-ECAO, and so Co-ECAO does not show the same substantial level of recovery of activity as does Co-HPAO under oxygen saturation (32). Another example of appreciable activity in a Co-substituted enzyme was presented for bovine serum amine oxidase (BSAO) by Suzuki et al (27), who reported 13.3% of native BSAO activity which corrects to 18.2% when allowance is made for the 73% Co content.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, as copper has been shown to be the most favorable metal for supporting catalysis in ECAO, it could be argued that this is consistent with a redox role involving a Cu(I)-TPQ SQ intermediate in line with recent evidence for PSAO and AGAO (38,39). However, an outer-sphere electron transfer mechanism would still be required to explain the observed catalytic activity in Co-ECAO, Co-BSAO (27), and Co-HPAO (32). Shepard et al suggested the possibility that an outer-sphere mechanism may operate for the Co- and Ni-substituted forms of AGAO (30).…”

Section: Discussionmentioning

confidence: 99%

Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase^,

et al. 2010

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To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only ∼12% wild-type activity as measured by kcat(amine). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60−90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.

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“…In addition, the two Cu ions exhibit a different reactivity since only one of them can be removed from BSAO with diethyldithiocarbamate in the presence of a reductant. The enzyme activity, strongly decreased in the half-Cu-depleted form and practically abolished in the fully Cu-depleted enzyme, is in part recovered by copper or cobalt reincorporation (Suzuki et al, 1981(Suzuki et al, , 1983Morpurgo et al, 1990). No differences in coordinated ligand structure between native and half-Cu-depleted BSAO could be detected by spin-echo experiments .…”

mentioning

confidence: 89%

Properties of cobalt‐substituted bovine serum amine oxidase

Agostinelli¹,

Morpurgo²,

Wang³

et al. 1994

European Journal of Biochemistry

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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.

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Preparation and Characterization of Cobalt(II)-Substituted Bovine Serum Amine Oxidase1

Cited by 14 publications

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Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase^,

Properties of cobalt‐substituted bovine serum amine oxidase

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Preparation and Characterization of Cobalt(II)-Substituted Bovine Serum Amine Oxidase1

Cited by 14 publications

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Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Reconstitution of Cu2+-depleted bovine serum amine oxidase with Co2+

Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase,

Properties of cobalt‐substituted bovine serum amine oxidase

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Exploring the Roles of the Metal Ions in Escherichia coli Copper Amine Oxidase^,