Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

Abstract

Tyrosinases mediate the ortho-hydroxylation and two-electron oxidation of monophenols to ortho-quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.

Activation of dioxygen by copper metalloproteins and insights from model complexes

“…This is consistent with the idea of an activated water molecule, along with conserved amino acid residues (glutamate and asparagine), playing a key role in the deprotonation of phenols during the catalytic cycle of Ty [168, 170]. …”

“…Additionally, it has been speculated that a highly conserved glutamate (in both Ty and CaOX) and an asparagine (conserved mainly in Ty) bind and activate a conserved water molecule towards deprotonation of phenols. Based on this observation, it has been proposed that an active-site asparagine is responsible for differentiating between monooxygenase and catechol oxidase activity [168–170]. …”

Immunological properties of oxygen-transport proteins: hemoglobin, hemocyanin and hemerythrin

“…These residues fix a conserved water molecule and lower its pK value to disrupt the hydrogen from the monophenols (i.e. deprotonate), so the resulting phenolate can bind to CuA to initiate the enzymatic cycle [159–161]. The proton will be bound by this water molecule to form a hydronium ion (H 3 O + ).…”

“…After release of the final product, namely o-quinone, a hydroxyl group bridges the two copper ions but will be discarded as a water molecule after obtaining a hydrogen back from the hydronium ion. Upon replacing either the asparagine or glutamine residues with different amino acids only catecholoxidase activity is possible [161]. In the absence of a true PO, chelicerates rely on the inducible PO activity of Hc as a substitute [28, 30, 162, 163, 203, 204].…”

Simplest Monodentate Imidazole Stabilization of the oxy‐Tyrosinase Cu2O2 Core: Phenolate Hydroxylation through a CuIII Intermediate

“…[21] A Cu(III) containing species coordinated by such biologically relevant ligands is unprecedented, and its characterization may provide insight regarding the active hydroxylating oxidant of tyrosinases. [4a, 22] …”