title: Identification of the copper-binding ligands of lysyl oxidase.
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 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.
authors.affiliation: Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA.
authors.forename: Karlo M
authors.initials: KM
authors.lastname: Lopez
authors.forename: Frederick T
authors.initials: FT
authors.lastname: Greenaway
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D008249:Protein-Lysine 6-Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D008024:Ligands; D006639:Histidine; D003300:Copper; D008249:Protein-Lysine 6-Oxidase