Evidence Consistent with the Requirement of Cresolase Activity for Suicide Inactivation of Tyrosinase

Abstract

Tyrosinase is a mono-oxygenase with a dinuclear copper catalytic center which is able to catalyze both the ortho-hydroxylation of monophenols (cresolase activity) and the oxidation of catechols (catecholase activity) yielding ortho-quinone products. Tyrosinases appear to have arisen early in evolution and are widespread in living organisms where they are involved in several processes, including antibiosis, adhesion of molluscs, the hardening of the exoskeleton of insects, and pigmentation. Tyrosinase is the principal enzyme of melanin formation in vertebrates and is of clinical interest because of the possible utilization of its activity for targeted treatment of malignant melanoma. Tyrosinase is characterised by an irreversible inactivation that occurs during the oxidation of catechols. In a recent publication we proposed a mechanism to account for this feature based on the orthohydroxylation of catecholic substrates, during which process Cu(II) is reduced to Cu(0) which no longer binds to the enzyme and is eliminated (reductive elimination). Since this process is dependent on cresolase activity of tyrosinase, a strong prediction of the proposed inactivation mechanism is that it will not be exhibited by enzymes lacking cresolase activity. We show that the catechol oxidase readily extracted from bananas (Musa cavendishii) is devoid of cresolase activity and that the kinetics of catechol oxidation do not exhibit inactivation. We also show that a species with the molecular mass of the putative cresolase oxidation product is formed during tyrosinase oxidation of 4-methylcatechol. The results presented are entirely consistent with our proposed mechanism to account for suicide-inactivation of tyrosinase.

Mechanistic studies of tyrosinase suicide inactivation

“…The absence of oxygenase activity was shown not to be due to lack of activation of the enzyme (Figure 2) or the presence of an inhibitor of oxygenase activity. 19 All these data are consistent with the requirement for oxygenase activity for suicide inactivation of tyrosinase and provide very strong support for the Quintox mechanism (Scheme 3). Further indirect evidence of the involvement of an oxygenase presentation in the inactivation mechanism (Scheme 3) is the inhibitory effect of addition of a monohydric phenol to the catechol oxidation mixture.…”

“…The behaviour of a catechol oxidase extracted from bananas (Musa cavendishii) indicates that, in contrast to tyrosinase, it oxidises a number of catecholic substrates without displaying suicide-inactivation kinetics. 19 Earlier results published by Padrón and co-workers 26 show firstorder kinetics exhibited by the banana enzyme which are claimed as evidence of enzyme inactivation. However, in this study 26 the exponential decrease in oxidation rate probably reflects the fall in oxygen concentration as shown by the reported polarimetric results.…”

“…This processing of catechols as phenols in the oxygenase cycle nicely accounts for the observed suicide-inactivation mechanism and we have described further experimental studies that support this mechanism. 19,20 Recognising the novelty and significance of our studies and proposals, another group has subsequently proposed a variation on the reductive-elimination theme to account for copper loss. 21 This alternative suggestion is summarised in Scheme 4.…”

“…Nevertheless, the product 12 has been identified by hplc- mass spectrometry in the case of 4-methylcatechol oxidation by tyrosinase and shown to be correlated with the loss of enzyme activity. 19 …”

Studies of the competing rates of catechol oxidation and suicide inactivation of tyrosinase

“…We have recently provided evidence that this inactivation occurs when the catecholic substrate presents itself to the active site as a phenolic substrate, [6][7][8][9] leading to irreversible reductive elimination of copper 10 from the enzyme. During the course of these studies we have examined a number of 4-substituted catecholic substrates and have measured rate constants for their oxidation (k 1 ) and for the rates at which they inactivate the enzyme (k 2 ) (Scheme 1).…”