Chemistry

Identification of the copper-binding ligands of lysyl oxidase

Document Type

Conference Proceeding

Abstract

In order to identify the ligands coordinating with copper in lysyl oxidase, the enzyme was expressed in an E. coli expression system and active enzyme obtained after refolding in the presence of Cu(II). The five histidines found in the putative copper-binding region were sequentially mutated to alanines and the enzymatic activities of the resultant mutants were monitored, together with the copper content, the CD and fluorescence spectra, and the redox-cycling activity. The spectroscopic results show that in all cases the protein folded correctly but that the coppercontent, enzymatic activity, and redox-cycling ability depended on the mutation. One mutant was fully functional, and two others completely lacked copper, the lysyl tyrosyl quinone (LTQ) cofactor, and activity. A fourth incorporated copper but lacked LTQ and enzymatic activity. The remaining mutant incorporated copper and had redox-cycling activity but no enzymatic activity. The results suggest that three of the five histidines in the putative copper-binding domain, H292, H294, H296, are the copper ligands and essential to the formation of LTQ. A fourth, H289, is not involved in LTQ formation or activity, while a fifth, H303, is suggested to be a general base in the catalytic mechanism. © Springer-Verlag 2010.

Publication Title

Journal of Neural Transmission

Publication Date

7-2011

Volume

118

Issue

7

First Page

1101

Last Page

1109

ISSN

0300-9564

DOI

10.1007/s00702-010-0559-4

Keywords

copper-containing amine oxidase, enzyme activity, fluorescence, Lysyl oxidase, site-directed mutagenesis

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