Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site.

Zeidan, Henry M.; Watanabe, Kojiro; Piette, Lawrence H.; Yasunobu, Kerry T.

doi:10.1016/s0021-9258(19)43874-x

Cited by 19 publications

(4 citation statements)

References 14 publications

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“…The final purified enzyme had a specific activity of 0.47 units/mg of protein. Protein concentration was determined from 280 nm using E 1cm 1% ) 20.8 (26) and a molecular weight of 172 000 (27). The BSAO protein was concentrated to 0.75 mM (1.5 mM TPQ) in 20 mM potassium phosphate (pH 7.0) in a Microcon 30 (Amicon) ultrafiltration device for all subsequent spectroscopic experiments.…”

Section: Methodsmentioning

confidence: 99%

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

et al. 1997

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Resonance Raman (RR) spectroscopy has proven to be an excellent technique for providing structural information about the 2,4, 5-trihydroxyphenylalaninequinone (TPQ) cofactor and for identifying the source of oxygen atoms during the posttranslational synthesis of the cofactor. Through specific labeling of the C2, C4, and C5 oxygens of TPQ in phenylethylamine oxidase (PEAO) from Arthrobacter globiformis, we have identified the C=O stretch of the C5 carbonyl at 1683 cm-1 (-27 in 18O) and the C=O stretch of the C2 carbonyl at 1575 cm-1 (-21 in 18O). These vibrational frequencies show that the C-O moiety at C5 has far greater double-bond character than at C2 or C4, thereby explaining the exclusive nucleophilic attack at the C5 position by substrates and substrate analogs. Bovine serum amine oxidase (BSAO) exhibits a similar nu(C=O) mode at 1678 cm-1 (-22 cm-1 in 18O). Aniline reacts with the TPQ cofactor of PEAO to form a new derivative (lambdamax at 450 nm) with properties similar to the proposed substrate-imine intermediate in the catalytic cycle. It retains the C2=O spectral features of the native enzyme and exhibits a new C5=N stretch at 1603 cm-1 (-29 in 15N). In contrast, methylamine reacts with both PEAO and BSAO under anaerobic conditions to form a different stable adduct (lambdamax at 385 nm) with properties closer to the proposed product-imine intermediate in the catalytic cycle. This species has a distinctive RR spectrum with a C=N stretch at 1617 cm-1 that corresponds to the atoms of the added methylamine (-58 cm-1 with CD3NH2, -19 cm-1 with CH315NH2). The lack of D2O dependence of nu(C=N) shows that this is a deprotonated imine, which would be more stable toward hydrolysis than the postulated protonated imine in the enzymatic reaction. However, the BSAO product imine (from methylamine) does undergo hydrolysis and conversion to semiquinone upon addition of cyanide. It is possible that the inactive form of the product imine is stabilized by deprotonation and flipping of the TPQ ring [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478].

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Section: Methodsmentioning

confidence: 99%

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

et al. 1997

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“…The purified enzyme was concentrated by using an Amicon cell and frozen. Protein concentrations were determined spectrophotometrically on either a Cary 118B or a Varian DMS 200 spectrophotometer at 280 nm using E\7°m = 20.8 (Zeidan et al, 1980) and a molecular weight of 170000 (Yasunobu et al, 1976). Although there has been some discrepency regarding the extinction coefficient and molecular weight of BSAO (Yamada & Yasunobu, 1962;Turini et al, 1982;Suzuki et al, 1983;Ishizaki & Yasunobu, 1980;Achee et al, 1968;Yasunobu et al, 1976), we find that (i) protein concentrations determined by Bio-Rad assay are consistent with those calculated from absorbance at 280 nm and (ii) the subunit molecular weight is clearly 85 000 on SDS gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer

Janes

Klinman²

1991

Biochemistry

124

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Recent evidence has shown that the active site cofactor in bovine serum amine oxidase (BSAO) is 2,4,5-trihydroxyphenylalanine or 6-hydroxydopa [Janes et al. (1990) Science 248, 981]. However, much ambiguity remains regarding the mechanism of the enzymatic reaction. Conflicting data exist for both the number of functional active sites in the dimeric enzyme and for the oxygen dependence of product release. To resolve these questions, a new method has been developed for the purification of BSAO which leads to the isolation of specific activity greater than or equal to 0.4 unit/mg of enzyme in 2-3 weeks. This highly active enzyme has been used to quantitate both aldehyde and ammonia release in the reductive half-reaction. Anaerobic incubation of enzyme and substrate resulted in the production of 2 mol of aldehyde/mol of enzyme, indicating the presence of a cofactor at each enzyme subunit. As anticipated for an aminotransferase reaction, no ammonia release was detected under comparable conditions. Active site titration of enzyme samples of varying specific activity with phenylhydrazine extrapolates to 1 mol of inhibitor/mol of enzyme subunit for BSAO of specific activity = 0.48 unit/mg. These findings contrast with numerous, previous reports of only one functional cofactor per enzyme dimer in copper amine oxidases.

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“…(KA McKeown, DMD, unpublished observations). Reports of a titratable cysteine associated with substrate reduction in other amine oxidases [14,15,28] may refer to residues that are not conserved in PSAO. The lack of a detectable free cysteine in PSAO would be rationalized by the formation of an intersubunit disulfide bond.…”

Section: Cysteine Residues Disulfide Bridgesmentioning

confidence: 99%

“…Structural details that should be of interest include the locations of cysteine residues and glycosylation sites. The cysteine residues in PSAO are of interest because several of them are conserved among eukaryotic but not prokaryotic amine oxidases, and structural or functional roles for these residues have been proposed [14,15]. Identifying the number and locations of glycosylation sites in PSAO may be important as all eukaryotic amine oxidases are glycoproteins.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 å resolution

et al. 1996

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There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

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Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site.

Cited by 19 publications

References 14 publications

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

Topaquinone-Dependent Amine Oxidases: Identification of Reaction Intermediates by Raman Spectroscopy

An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 å resolution

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