1
142
Vol. 36, No. 7
RESULTS AND DISCUSSION
Table 1. Percentage Inhibition of Monophenolase and Diphenolase Ac-
tivities of Polyphenol Oxidase in the Presence of 5 or 6
When attempts are made to characterize a monophenol as
possible substrate for PPO, problems may arise—for example,
no enzymatic activity is detected or, if the monophenol is
assayed by measuring the enzyme activity on l-tyrosine or
l-DOPA and following the increase in absorbance caused by
the formation of dopachrome at a wavelength of 475nm, the
dopachrome formation rate usually diminishes since most
monophenols, including 5, give rise to o-quinones with low
molar absorptivity coefficients and so, in its presence, the
a)
b)
Concentration Monophenolase
Diphenolase
Substrates
(mm)
activity (%)
activity (%)
5
0.1
52.9
74.3
82.9
81.2
84.6
86.3
10.8
35.6
49.5
63.4
79.3
86.8
0
0
.3
.5
6
0.1
0
0
.3
.5
a) Monophenolase activity, the experimental conditions were: phosphate buffer
formation of dopachrome is slowed, and, consequently, such 30mm, pHꢀ6.0 at 25°C, [E] ꢀ50nm, [l-tyrosine] ꢀ0.26mm, [l-DOPA] /[l-tyro-
0
0
0
sine] ꢀ0.046. Umbelliferone, (5) (mm): 0.1, 0.3 and 0.5. Esculetin, (6) (mm): 0.1, 0.2
0
monophenols are usually classified as inhibitors.
and 0.5. b) Diphenolase activity, the experimental conditions were: phosphate buffer
When studying the action of 5 and 6 on PPO activity by 30mm, pHꢀ6.0 at 25°C, [E] ꢀ15nm, [l-DOPA] ꢀ0.5mm. Umbelliferone (5) and es-
0
0
culetin (6), the same concentrations as used for monophenolase activity.
measuring the monophenolase and diphenolase activities on l-
tyrosine or l-DOPA, the effects described above normally lead
to them being described as powerful inhibitors of PPO: 6 with
6
)
an IC ꢀ0.043mm when assayed with l-DOPA as substrate,
5
0
and 5 with a K ꢀ0.014mm when assayed with l-tyrosine as
I
4)
substrate. Moreover, the characterization of possible ty-
rosinase substrates is complicated by the fact that the reaction
product (an o-quinone) is usually unstable.
21,25,26)
In a previous work using a chronometric method,
we
characterized 6 not as a PPO inhibitor but as a true substrate
4
)
of the same, a conclusion also reached by other authors.
4)
However, 5 has been classified as an inhibitor, a fact that can
be explained if we take into consideration the enzyme’s action
mechanism depicted in Chart 1 (see below).
Behaviour of 5 and 6 as Apparent Inhibitors of the Ac-
tivity of PPO on l-Tyrosine and l-DOPA Table 1 shows
the apparent % inhibition of PPO activity exercised by 5 and
6
on the monophenolase and diphenolase of PPO when mea-
sured with l-tyrosine or l-DOPA, respectively. When the inhi-
bition of PPO by 6 is studied, this substrate apparently inhibits
both the activity on l-tyrosine and l-DOPA more strongly,
because it is a better substrate for the enzyme than 5. How- Fig. 1. Spectrophotometric Recordings of 6 and 5 at Different pH Val-
ever, the inhibition of PPO by a monophenol, like 5, or by an ues
The experimental conditions for 6 were: [esculetin] ꢀ0.1 mm, phosphate buffer
o-diphenol, like 6, is greater in the case of the monophenolase
activity of PPO on l-tyrosine, since in this case the enzyme
0
3
(
0mm, at 25°C, at values pH: 8.3 (a), 7.9 (b), 7.3 (c), 7.1 (d), 6.6 (e), 6.0 (f) and 5.5
g) and acetate buffer 30mm at pH values of 5.3 (h) and 4.5 (i). Inset. The experi-
is partly inhibited by the l-tyrosine itself (see Chart 1). More- mental conditions for 5 were [umbelliferone]
ꢀ0.1 mm and the same pH-values as
0
in Fig. 2.
over, the binding of the monophenols to the met form is strong
2
7)
since it is facilitated by a base probably a histidine.
Physico-Chemical Characteristics of 5 and 6 The does no accumulate o-diphenol in the medium (k ꢀ0, Chart
10
monophenol 5 has a pK value of 6.89, which is low compared 1), as occurs in the case of the o-dopaquinone, and it is neces-
a
with other monophenols, and this is similarly the case with 6 sary to add a small quantity of o-diphenol for the system to
(
pK ꢀ7.64). The existence of this pK is confirmed in Fig. 1 show activity. When the oxygen consumption rate is measured
a
a
and Fig. 1 inset, by the appearance of the isosbestic points. in the action of PPO on 5, by adding 6 at a constant ratio (R),
Furthermore, the high chemical shift values (δ) for the carbon Rꢀ[esculetin] /[umbelliferone] ꢀ0.02, a rate in oxygen con-
atoms supporting the hydroxyl group (δ ꢀ158.1ppm for 5 and sumption was observed (results not shown). The dependence
2
8)
0
0
1
δ ꢀ146.9ppm and δ ꢀ144ppm for 6) suggests that both are of the oxygen consumption rate on the concentration of um-
1
2
very bad substrates for PPO, as will be confirmed below.
belliferone was hyperbolic. Nonlinear analysis of these data
Considerations on the PPO Action Mechanism The according the Michaelis equation gives the values of k and
cat
−1
kinetic mechanism of PPO in its activity on monophenols and K as 0.09±0.02s and 0.17±0.06mm, respectively.
m
o-diphenols is described in Chart 1, where it can be seen that
To confirm that the product of PPO acting on 5 is 6 (Fig. 2),
for PPO to act on a monophenol the presence of an o-diphenol PPO was made to react with 5 in the presence of ascorbic acid
is necessary. In the case of monophenols that give rise to un- in different conditions: Fig. 2a (◆) with catalytic quantities of
stable o-quinones (for example o-dopaquinone) that can accu- 6, Fig. 2b (▲) with catalytic quantities of TBC, and Fig. 2c
mulate o-diphenol in the medium as they evolve, the enzyme (○) in the absence of o-diphenol. At given times, an aliquot
reaches the steady state after a lag period, which is the time was oxidized by periodate in excess and the absorbance cor-
the system needs to accumulate the o-diphenol concentration responding to the o-quinone of 6 was monitored (see Fig. 2).
2
8)
to reach the steady state. In the case of 5, the o-quinone As can be seen, the absorbance increased with the incubation