The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques.
The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3+/-0.1 compared to 3.3+/-0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.
Bukh
Christian
C
Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Bjerrum
Morten J
MJ
D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D003302:Coprinus / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013057:Spectrum Analysis / Q000379:methods*
D005656:Fungal Proteins; D003300:Copper; D042845:Laccase