Mechanistic Insight into the Activity of Tyrosinase from Variable‐Temperature Studies in an Aqueous/Organic Solvent

Abstract

The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4% methanol-glycerol (7:1, v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61+/-9 kJ mol(-1) irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10% to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein-substrate interactions. The substrate 4-hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH(double dagger) approximately = 150 kJ mol(-1) and deltaS(double dagger) approximately = 280 J K(-1) mol(-1), due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.

Endogenous Arene Hydroxylation Promoted by Copper(I) Cluster Helicates

“…The kinetic studies for the hydroxylation reaction showed a DH°v alue of around 70 kJ mol À1 , which is similar to those found for the hydroxylations mediated by tyrosinase. [27] We believe that this approach could open new perspectives for the design of functional helicates with catalytic activity.…”

Similar enzyme activation and catalysis in hemocyanins and tyrosinases

“…Concomitant with attack on the aromatic ring the O-O bond is cleaved (Decker and Tuczek, 2000;Decker et al, 2001aDecker et al, , 2006. The mechanistic proposal of concomitant attack on the phenol aromatic ring and O-O bond cleavage in the monophenolase reaction by tyrosinase (Scheme 1) has recently gained experimental support by a study of kinetic isotope effects (Granata et al, 2006).…”

Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η1:η1‐O2‐Peroxo Dicopper(II) Complex

“…[13,18] For instance, crystallographic analyses show that the oxygenated form of tyrosinase (oxyTyr) binds a molecule of the phenolic substrate in one of the copper centers, [9] triggering a rotation of the peroxo core and orientating the ortho-phenolic site into a position that is conducive for electrophilic attack by the Cu 2 -O 2 center (Scheme 1). [9,15,19] Beyond its biological relevance, the topic of O 2 activation at dicopper sites has elements of fundamental chemical interest and significance. From a very basic point of view, it involves the use of inexpensive, abundant, …”

“…[c] See Granata et al [19] [d] See Palavicini et al [34] [e] See Itoh et al [35] [f] See Mirica et al [37] [g] See Company et al [38] www.chemeurj.org entropic energetically nonfavorable organization of the system. On the other hand, activation values for the phenolate oxidation process derived from the Eyring plot are…”