12
1 Melanin Chemistry
contained in DCT are zinc ions [45, 46]. Solano and colleagues analyzed the composition of metal ions by atomic absorption spectroscopy, and showed that DCT
contained almost no copper or iron ions, but it contained zinc ions. Furthermore, after
removing metal ions using cyanides (or other chelating agents) and then recombining
with various metal ions, zinc ions showed the largest restoration of the enzymatic
activity of DCT. Unlike copper ions, zinc ions hardly cause oxidations, indicating
the markedly different function of DCT.
1.3.4 Reinvestigation of Tyrosinase Actions
The existence of the induction period of tyrosinase and its shortening by dopa have
long been unresolved. This puzzle has gradually become resolved as melanogenesis
has been investigated in more detail. Tyrosinase has long been known as a “copper
protein” containing two copper ions, which are responsible for the oxidation reaction [27]. A study of the crystal structure of mushroom tyrosinase [47] showed the
structure of the active site containing copper ions.
At the active site of tyrosinase, there is a pair of copper ions, each coordinated with
three nitrogen atoms in histidine residues, and the pair center is able to bind oxygen
and hydroxyl ions. The formation of a catechol (dopa) from a monophenol (tyrosine) in melanogenesis indicates that tyrosinase should have an ability to uptake and
transfer oxygen (monooxygenase activity). Considering that tyrosinase-catalyzed
oxidation consumes oxygen in the air [30], tyrosinase must be able to uptake oxygen
from the air again after the reaction to restore the catalytic activity.
Most of the isolated tyrosinase is present in a state called met-tyrosinase form,
in which Cu(II) ions are combined with a hydroxyl ion. This form cannot uptake
oxygen any more because there are no oxidation states higher than Cu (II). However,
when the coppers are reduced to Cu (I) by reducing agents to release the bound
hydroxyl ion (deoxy-tyrosinase form), it becomes available for oxygen uptake in the
peroxide state (oxy-tyrosinase form) from the air. Furthermore, as the fourth form, an
irreversibly inactivated deact-tyrosinase form is generated when the oxy-tyrosinase
acted on oxidation of catechols or resorcinols (Fig. 1.5) [48]. It has been hypothesized
that there may be some connection between the mechanism to re-uptake oxygens and
the mechanism by which dopa shortens the induction period of tyrosinase.
The shortening of the induction period can be explained by considering that dopa,
resulting from tyrosine oxidation, acts as a reducing agent, thereby reducing the
copper ions of tyrosinase so that it can react with oxygen again. To demonstrate this
mechanism, the amount of oxygen uptake was investigated. When 4-hydroxyanisole
was used as a substrate for tyrosinase, an equimolar O 2 consumption with respect to
the substrate was observed. In contrast, when tyrosine was used as a substrate, the O 2
consumption was 1.5-fold higher than the amount of substrate [49]. This is because
dopa, resulting from tyrosine oxidation, is further oxidized by tyrosinase to consume
more oxygen. This extra O 2 consumption (0.5-fold amount of the substrate) can be
regarded as a stoichiometry of 2 mol of dopa to 1 mol of O 2 . This stoichiometric ratio
1 Melanin Chemistry
contained in DCT are zinc ions [45, 46]. Solano and colleagues analyzed the composition of metal ions by atomic absorption spectroscopy, and showed that DCT
contained almost no copper or iron ions, but it contained zinc ions. Furthermore, after
removing metal ions using cyanides (or other chelating agents) and then recombining
with various metal ions, zinc ions showed the largest restoration of the enzymatic
activity of DCT. Unlike copper ions, zinc ions hardly cause oxidations, indicating
the markedly different function of DCT.
1.3.4 Reinvestigation of Tyrosinase Actions
The existence of the induction period of tyrosinase and its shortening by dopa have
long been unresolved. This puzzle has gradually become resolved as melanogenesis
has been investigated in more detail. Tyrosinase has long been known as a “copper
protein” containing two copper ions, which are responsible for the oxidation reaction [27]. A study of the crystal structure of mushroom tyrosinase [47] showed the
structure of the active site containing copper ions.
At the active site of tyrosinase, there is a pair of copper ions, each coordinated with
three nitrogen atoms in histidine residues, and the pair center is able to bind oxygen
and hydroxyl ions. The formation of a catechol (dopa) from a monophenol (tyrosine) in melanogenesis indicates that tyrosinase should have an ability to uptake and
transfer oxygen (monooxygenase activity). Considering that tyrosinase-catalyzed
oxidation consumes oxygen in the air [30], tyrosinase must be able to uptake oxygen
from the air again after the reaction to restore the catalytic activity.
Most of the isolated tyrosinase is present in a state called met-tyrosinase form,
in which Cu(II) ions are combined with a hydroxyl ion. This form cannot uptake
oxygen any more because there are no oxidation states higher than Cu (II). However,
when the coppers are reduced to Cu (I) by reducing agents to release the bound
hydroxyl ion (deoxy-tyrosinase form), it becomes available for oxygen uptake in the
peroxide state (oxy-tyrosinase form) from the air. Furthermore, as the fourth form, an
irreversibly inactivated deact-tyrosinase form is generated when the oxy-tyrosinase
acted on oxidation of catechols or resorcinols (Fig. 1.5) [48]. It has been hypothesized
that there may be some connection between the mechanism to re-uptake oxygens and
the mechanism by which dopa shortens the induction period of tyrosinase.
The shortening of the induction period can be explained by considering that dopa,
resulting from tyrosine oxidation, acts as a reducing agent, thereby reducing the
copper ions of tyrosinase so that it can react with oxygen again. To demonstrate this
mechanism, the amount of oxygen uptake was investigated. When 4-hydroxyanisole
was used as a substrate for tyrosinase, an equimolar O 2 consumption with respect to
the substrate was observed. In contrast, when tyrosine was used as a substrate, the O 2
consumption was 1.5-fold higher than the amount of substrate [49]. This is because
dopa, resulting from tyrosine oxidation, is further oxidized by tyrosinase to consume
more oxygen. This extra O 2 consumption (0.5-fold amount of the substrate) can be
regarded as a stoichiometry of 2 mol of dopa to 1 mol of O 2 . This stoichiometric ratio