Topaquinone-dependent amine oxidases: Identification of reaction intermediates by Raman spectroscopy

Nobuhumi Nakamura, Pierre Moenne-Loccoz, Katsuyuki Tanizawa, Minae Mure, Shinnichiro Suzuki, Judith P. Klinman, Joann Sanders-Loehr

Research output: Contribution to journalArticle

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Abstract

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 v(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 (λ(max) 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 (λ(max) 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 CH3 15NH2). The lack of D2O dependence of v(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.

Original languageEnglish (US)
Pages (from-to)11479-11486
Number of pages8
JournalBiochemistry
Volume36
Issue number38
DOIs
StatePublished - Sep 23 1997

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Reaction intermediates
Imines
Raman Spectrum Analysis
Amines
Raman spectroscopy
Oxidoreductases
Hydrolysis
Substrates
Serum
Oxygen
Arthrobacter
Atoms
Deprotonation
Cyanides
Vibrational spectra
Labeling
6-hydroxydopa quinone
Raman scattering
Derivatives
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nakamura, N., Moenne-Loccoz, P., Tanizawa, K., Mure, M., Suzuki, S., Klinman, J. P., & Sanders-Loehr, J. (1997). Topaquinone-dependent amine oxidases: Identification of reaction intermediates by Raman spectroscopy. Biochemistry, 36(38), 11479-11486. https://doi.org/10.1021/bi9708139

Topaquinone-dependent amine oxidases : Identification of reaction intermediates by Raman spectroscopy. / Nakamura, Nobuhumi; Moenne-Loccoz, Pierre; Tanizawa, Katsuyuki; Mure, Minae; Suzuki, Shinnichiro; Klinman, Judith P.; Sanders-Loehr, Joann.

In: Biochemistry, Vol. 36, No. 38, 23.09.1997, p. 11479-11486.

Research output: Contribution to journalArticle

Nakamura, N, Moenne-Loccoz, P, Tanizawa, K, Mure, M, Suzuki, S, Klinman, JP & Sanders-Loehr, J 1997, 'Topaquinone-dependent amine oxidases: Identification of reaction intermediates by Raman spectroscopy', Biochemistry, vol. 36, no. 38, pp. 11479-11486. https://doi.org/10.1021/bi9708139
Nakamura, Nobuhumi ; Moenne-Loccoz, Pierre ; Tanizawa, Katsuyuki ; Mure, Minae ; Suzuki, Shinnichiro ; Klinman, Judith P. ; Sanders-Loehr, Joann. / Topaquinone-dependent amine oxidases : Identification of reaction intermediates by Raman spectroscopy. In: Biochemistry. 1997 ; Vol. 36, No. 38. pp. 11479-11486.
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N2 - 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 v(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 (λ(max) 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 (λ(max) 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 CH3 15NH2). The lack of D2O dependence of v(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.

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