caa a22 4500 445376465 CHVBK 20180317143004.0 cr unu---uuuuu 170323e20110701xx s 000 0 eng 10.1007/s00702-010-0559-4 doi (NATIONALLICENCE)springer-10.1007/s00702-010-0559-4 Identification of the copper-binding ligands of lysyl oxidase [Elektronische Daten] [Karlo Lopez, Frederick Greenaway] 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 copper-content, 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 Lysyl oxidase nationallicence Copper-containing amine oxidase nationallicence Site-directed mutagenesis nationallicence Fluorescence nationallicence Enzyme activity nationallicence BAPN : β-aminopropionitrile nationallicence BNPS-skatole : 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine nationallicence EEM : Excitation-emission matrix 2D fluorescence spectroscopy nationallicence IPTG : Isopropyl-β-d-thiogalactoside nationallicence LOX : Lysyl oxidase nationallicence LTQ : Lysyl tyrosyl quinone nationallicence proLOX : Prolysyl oxidase nationallicence LOXL : Lysyl oxidase-like protein nationallicence TPQ : Topaquinone nationallicence Lopez Karlo Carlson School of Chemistry and Biochemistry, Clark University, 01610, Worcester, MA, USA aut Greenaway Frederick Carlson School of Chemistry and Biochemistry, Clark University, 01610, Worcester, MA, USA aut Journal of Neural Transmission Springer Vienna 118/7(2011-07-01), 1101-1109 0300-9564 118:7<1101 2011 118 702 https://doi.org/10.1007/s00702-010-0559-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00702-010-0559-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lopez Karlo Carlson School of Chemistry and Biochemistry, Clark University, 01610, Worcester, MA, USA aut NATIONALLICENCE 700 1- Greenaway Frederick Carlson School of Chemistry and Biochemistry, Clark University, 01610, Worcester, MA, USA aut NATIONALLICENCE 773 0- Journal of Neural Transmission Springer Vienna 118/7(2011-07-01), 1101-1109 0300-9564 118:7<1101 2011 118 702 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer