Bovine serum amine oxidase

In enzyme catalysis, 249, 373-397 [bovine serum amine oxidase, 249, 393-394 coupled motion and, 249, 386-388 demonstration, 249, 374-386 (breakdown of rule of geometric mean in,... 

Su Q. Klinman J. P. Probing the mechanism of proton coupled electron transfer to dioxygen the oxidative half-reaction of bovine serum amine oxidase. Biochemistry 1998, 37, 12513-12525. 

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

The Bruno and Bialik, (1992) theory which takes into account nuclear tunneling (Section 4.2.1), was applied to an analysis of anomalous Schaad-Swain exponents in a reaction catalyzed by bovine serum amine oxidase, BSAO (Grant and Klinman, 1989). The isotope effect in this reaction is found to be markedly larger than one, expected classically. Theoretical values of H/T and D/T KIFs and its temperature dependence match Grant and Klinman s experimental data. 

Stopped flow kinetic studies by Hartmann and Klinman (1991) on bovine serum amine oxidase and by Olsson et al. (1976) on pig plasma amine oxidase showed that the substrate Schiff-base has an absorbance maximum at 350nm. 

DeBiase, D., Agostinelli, E., DeMatteis, G., Mondovi, B., and Morpurgo, L., 1996, Half of the sites reactivity of bovine serum amine oxidase reactivity and chemical identity of the second site, Eur. J. Biochem. 237 7279732. 

Grant, K. L., and Klinman, J. P., 1989, Evidence that both protium and deuterium undergo significant tunneling in the reaction catalysed by bovine serum amine oxidase. Biochemistry 28 6597n6605. 

Suzuki, S., Sakurai, T., Nakahara, A., Manabe, T., and Okuyama, T., 1986, Roles of the two copper ions in bovine serum amine oxidase. Biochemistry 25 338n341. 

Unlike bovine serum amine oxidase, which is available in gram quantities and was used as the prototypic system for the establishment of TPQ, a typical preparation of LO yields about 5 mg (starting with about 500 grams of aorta tissue). In an effort to minimize protein loss, purification was stopped after the stage where LO (32 kDa) coelutes with a second protein (24 kDa) from Sephacryl S-200 gel filtration. The two-banded protein was labeled with [ 4C]phenyl-hydrazine, to yield the expected chromophore for a phenylhydrazone derivative of a quinone structure (Fig. 1). Analysis of the i C-labeled protein by SDS gel electrophoresis showed almost exclusive incoiporation of into the 32-kDa LO band (13). Therefore, the unreactive 24-kDa band would not affect subsequent procedures since selection of active site-derived peptides from LO was dependent on screening for in addition to monitoring die UV-Vis absorption of the newly formed chromophore. 

Bovine serum amine oxidase (BSAO) is a copper-containing amine oxidase which utilizes a covalently bound 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor in the two-electron oxidation of a broad spectrum of primary amines [99]. The oxidation is thought to proceed via the formation of an iminium complex between the oxidized form of the cofactor and the primary amine (1 in Scheme 10.4). The substrate imine undergoes deprotonation to form product imine, which, after hydrolysis, releases aldehyde product and reduced cofactor [100]. Proton transfer is either partially or largely rate-limiting for the oxidation of benzylamines, as evidenced by a large deuterium isotope effect at the methylene adjacent to the amino group [36, 101, 102]. 

Structure of the active center. The active centers of this dimeric enzyme are so well embedded into its protein structure that they are inaccessible to the solvent. The two centers are situated approximately 30 A apart from each other but connected by /3-strands. The active center consists of a type 2 copper center and a cofactor. Sequence comparisons have established that the residues His 8, His 246, and His 357 coordinate the copper ions in both yeast and plants (e.g., lentil seeds) [120,122]. The participating cofactor is typical for amine oxidases, diamine oxidases, and lysyl oxidases but has not yet been found in any other protein - 2,4,5-trihydroxy-phenylalanine quinone [123, 124] (also known as TOPA-quinone, TPQ or 6-hydroxy-DOPA quinone), an internal cofactor which is created by post-translational modification of the tyrosine in position 387 [120]. The consensus sequence of the amino acids neighboring the TOPA cofactor are conserved in all known amine oxidases - Asn-TOPA-Asp/Glu [113,120, 123,125-127]. The positions of the histidine ligands relative to TOPA quinone are conserved in all known amine oxidases as well. The chain lengths of the amine oxidase monomers vary according to the organism of origin 692 residues in yeast [128], 762 in bovine serum amine oxidase [128,129] and 569 in the enzyme from lentil seeds [120,130]. 

The crystal structure of CuAO has been solved from Escherichia coli (ECAO), pea seedling (PSAO), Arthrobacter globiformis (AGAO), Hansetmla polymorpha (HPAO), Pichia pastoris (PPLO), " bovine serum amine oxidase (BSAO), ° and human vascular adhesion protein (VAP-1). ... 

The copper can be reversibly removed from the active site by reaction with diethyldithiocarbamate under non-reducing conditions [86-88], or by cyanide after reduction by dithionate to Cu(I) [78]. The catalytic activity of the enzyme can be restored with high yield by addition of free Cu(II) ions to the apoenzyme [78,86]. In the case of pea seedling amine oxidase, addition of other bivalent metal ions does not lead to reactivation [86]. However the activity can be partially restored (from 15%) for the amine oxidase from bovine plasma by adding Co(II) [89]. Addition of Co(II) and Ni(II) can restore the original spectrum of the native enzyme with bovine serum amine oxidase reduced by dithionate [90]. 

Copper-depleted bovine serum amine oxidase has been recently reconstituted with Co(II) ions. The benzylamine oxidase activity of the enzyme was increased to 20% on incorporation of cobalt. Furthermore, Co(II) restored to nearly native level the intensity of the absorption spectrum and the reactions with phenylhydrazine or benzylhydrazine, which had been slowed down or abolished, respectively, in Cu(II)-depleted samples. The amine oxidase activity of the Co(II)-derivative, which cannot form a semiquinone radical as an intermediate of the... 

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