Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.
Ключові слова
Анотація
In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies