Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

Area of Science:

• Biochemistry
• Enzymology
• Protein Science

Background:

• Tyrosinases and catechol oxidases are type 3 copper proteins with distinct enzymatic activities.
• Tyrosinases hydroxylate monophenols, while catechol oxidases oxidize diphenols.
• The molecular basis for this functional discrimination has remained largely unexplained.

Purpose of the Study:

• To investigate the structural and functional basis for the difference between tyrosinase and catechol oxidase activity.
• To determine the role of specific amino acid residues, particularly asparagine and glutamate, in tyrosinase function.

Main Methods:

• Site-directed mutagenesis was employed to introduce an asparagine residue into a diphenolase-active polyphenoloxidase.
• Enzyme activity assays were performed to compare the substrate specificity of the wild-type and mutated enzymes.
• Structural analysis focused on the role of the asparagine and glutamate in orienting a conserved water molecule.

Main Results:

• A polyphenoloxidase with only diphenolase activity was successfully converted to a tyrosinase by introducing an asparagine residue.
• The presence of both asparagine and a conserved glutamate is essential for orienting a water molecule to abstract a proton from monophenols.
• This provides direct evidence for a proton shuttle mechanism in tyrosinase activity.

Conclusions:

• The asparagine residue, in conjunction with a conserved glutamate, is crucial for the proton abstraction step in monophenol hydroxylation.
• This finding elucidates the mechanism underlying the functional difference between tyrosinases and catechol oxidases.
• The study establishes a proton shuttle as a key determinant of tyrosinase activity in type 3 copper proteins.