IDENTIFICATION OF THE VERATRYL ALCOHOL BINDING-SITE IN LIGNIN PEROXIDASE BY SITE-DIRECTED MUTAGENESIS

Site-directed mutagenesis was used to identify the veratryI alcohol bi nding site of lignin peroxidase. The cDNA encoding isozyme H8 was muta ted at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat i s decreased for both mutants of Glu146. The reactivity of mutants (kca t/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the m utants for veratryl alcohol were decreased by at least half. The oxida tion of guaiacol by these mutants were more significantly affected. Th ese results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by Lignin peroxidase. (C) 1998 Academic P ress.