In recent years evidence has been accumulating that many enzymes proceed through radical mechanisms. Most reactions involve redox cofactors for which radical intermediates are plausible based on model studies of cofactor chemistry. New types of mechanisms have been proposed recently involving radicals on amino acid side chains. Although there has been much compelling indirect evidence consistent with H-atom abstraction from substrates by protein-derived radicals, to date there has been no direct evidence. Galactose oxidase from the fungus Dactylium dendroides catalyzes the oxidation of primary alcohols with O2, producing aldehydes and H2O2. Based on recent EPR, resonance Raman, and X-ray crystallographic structural information and on extensive preliminary studies with mechanism-based inactivating substrate analogs, a new mechanism for galactose oxidase catalysis is proposed featuring rate-determining H-atom abstraction by a tyrosine radical from the alcohol CH2. The reaction mechanism of galactose oxidase catalysis will be studied in the proposal research. The resulting detailed reaction mechanism should become a paradigm for substrate activation using protein-centered radicals for H- atom abstraction from substrates. Such fundamental information will be useful in understanding more biomedically relevant enzymes, such as ribonucleotide reductases, which are more difficult to study due to stringent substrate specificities.
