Copper amine oxidases are homodimeric enzymes containing one Cu2+ ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5–9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (Kb) against pH is bell-shaped, indicating two pKas of 5.8 and ∼8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the Kb versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.
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August 2002
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Research Article|
August 01 2002
Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue
Colin G. SAYSELL;
Colin G. SAYSELL
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Winston S. TAMBYRAJAH;
Winston S. TAMBYRAJAH
1
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Jeremy M. MURRAY;
Jeremy M. MURRAY
2
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Carrie M. WILMOT;
Carrie M. WILMOT
3
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Simon E.V. PHILLIPS;
Simon E.V. PHILLIPS
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Michael J. McPHERSON;
Michael J. McPHERSON
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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Peter F. KNOWLES
Peter F. KNOWLES
4
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
4To whom correspondence should be addressed (e-mail p.f.knowles@leeds.ac.uk).
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Publisher: Portland Press Ltd
Received:
October 05 2001
Revision Received:
March 01 2002
Accepted:
May 02 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 365 (3): 809–816.
Article history
Received:
October 05 2001
Revision Received:
March 01 2002
Accepted:
May 02 2002
Citation
Colin G. SAYSELL, Winston S. TAMBYRAJAH, Jeremy M. MURRAY, Carrie M. WILMOT, Simon E.V. PHILLIPS, Michael J. McPHERSON, Peter F. KNOWLES; Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue. Biochem J 1 August 2002; 365 (3): 809–816. doi: https://doi.org/10.1042/bj20011435
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