Galactose Oxidase Background
Over the past 20 years, there has been a growing appreciation for the catalytic utility of protein-derived free radical cofactors in enzymes. Free radical chemistry is harnessed to catalyze bond activation and molecular rearrangements in a wide variety of enzymes, including ribonucleotide reductase, DNA photolyase, cytochrome c peroxidase, pyruvate-formate lyase, lysine-2,3-aminomutase, prostaglandin H synthase and glyoxal oxidase. It is becoming increasingly clear that the environment of a protein-based radical can play a significant role in modulating its properties and reactivity. Substantial evidence indicates that second coordination shell effects in metalloproteins may profoundly influence the reactivity and electronic structure of active site metal ions, while weak interactions (such as hydrogen bonds) have been strategically manipulated to control reactivity in proteins and model complexes.
Crystal structure shows a distorted square pyramidal Cu(II) coordination geometry with an axial Y495 ligand and four equatorial ligands of H496, H581, H 2O (pH 7.0) or acetate (pH 4.5), and the unique crosslinked Tyr-Cys cofactor. W290 is stacking over Tys-Cys cofactor in the active site.
GO catalyzes 2-electron oxidation of a wide range of primary alcohols to aldehydes with the concomitant reduction of oxygen to hydrogen peroxide.
The modified tyrosine residue is believed to be the second redox site during the catalytic cycle (see proposed catalytic cycles).
Galactose oxidase displays three distinct oxidation states: oxidized [Cu(II)-Y•]; semireduced [Cu(II)-Y]; and reduced [Cu(I)-Y]. Only the [Cu(II)-Y•] and the [Cu(I)-Y] states are involved in the catalytic cycle, whose structures have not yet been fully characterized. As described above, the second coordination sphere residues may have significant effects in the structure of these distinct oxidation states.
Galactose oxidase also undergoes post-translational processing, involving cleavage of a 17 amino acid pro-sequence and the cross-linking of C228 and Y272 to form the redox cofactor of the active mature enzyme. Cleavage of pro-sequence requires Cu and O 2 but its role in biogenesis is not yet clear. The pro-sequence is not absolutely required but it may enhance the rate of cofactor formation.
There are numerous examples of the utilization of GO in biosensor, chemical synthesis, and diagnostics. Sensor incorporating GO have been used to measure D-galactose, lactose and other GO substrate concentrations in process monitoring blood samples and quality control in dairy industries. Enzymatic synthesis of carbohydrates by GO circumvents the requirement of protecting the hydroxyl group. GO catalyzes the oxidation of cell surface polysaccharides and is an essential step in the radiolabeling of membrane bound glycoproteins. The enzyme can also be used to detect a disaccharide tumor marker in colon cancer and pre-cancerous condition.
Research Focus in the Lab
We are interested in exploring the role of the second coordination sphere residues on the reactivity and stability of Tyr-Cys radical cofactor, the catalytic reaction in GO and its Cu geometry using both experimental and theoretical approaches. We also plan to investigate the hypothesis that the pro-sequence may play an important role in the biogenesis of the Tyr-Cys cofactor of galactose oxidase.