Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies

J Biol Inorg Chem. 2006 Jun;11(4):514-26. doi: 10.1007/s00775-006-0102-0. Epub 2006 Apr 21.


Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E0 =455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the kcat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15-0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacillus subtilis / enzymology*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites / genetics
  • Copper / chemistry*
  • Copper / metabolism
  • Enzyme Stability / genetics
  • Laccase / chemistry*
  • Laccase / genetics
  • Laccase / metabolism
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Protein Binding / genetics
  • Protein Structure, Tertiary / genetics
  • Structure-Activity Relationship
  • Thermodynamics


  • Bacterial Proteins
  • Mutant Proteins
  • Copper
  • Laccase