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Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue
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posted on 2023-06-08, 09:48 authored by Colin G Saysell, Winston S Tambyrajah, Jeremy M Murray, Carrie M Wilmot, Simon E V Phillips, Michael J McPherson, Peter F KnowlesCopper amine oxidases are homodimeric enzymes containing one Cu(2+) 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 (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 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 K(b) 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
History
Publication status
- Published
Journal
Biochemical JournalISSN
0264-6021Publisher
Portland PressPublisher URL
External DOI
Volume
365Page range
809-816Department affiliated with
- Biochemistry Publications
Notes
I and Dr C.G. Saysell contributed the majority of the results published in this paper. Others in the paper were in the group and collaborators with our Prof. M.J. McPherson and Prof. P.F. Knowles groupFull text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
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