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Electronic Supplementary Material
For
Activation Mechanism of MelB Tyrosinase from Aspergillus oryzae
by Acidic Treatment
Nobutaka Fujieda･Michiaki Murata･Shintaro Yabuta･Takuya Ikeda･Chizu Shimokawa･Yukihiro Nakamura･Yoji Hata･Shinobu Itoh
N. Fujieda･M. Murata･S. Yabuta･T. Ikeda･C. Shimokawa･S. Itoh
Department of Material and Life Science,
Division of Advanced Science and Biotechnology,
Graduate School of Engineering,
Osaka University,
2-1 Yamada-oka,
Suita, Osaka 565-0871, Japan
e-mail: S.I.: [email protected], N.F.: [email protected]
Yukihiro Nakamura･Yoji Hata
Research Institute Gekkeikan Sake Co., Ltd,
101 Shimotoba-koyanagi-cho,
Fushimi-ku, Kyoto, 612-8385, Japan
2
Contents
Figure S1 Time courses of the absorption change at 475 nm for the reaction of tyrosinase with
L-DOPA and with L-tyrosine Figure S2 Michaelis-Menten plot Figure S3 The gelfiltration chromatography of the pro-tyrosinase and activated tyrosinase Figure S4 HPLC elution diagram of the final reaction mixture of trypsin treated tyrosinase and
4-methylphenol Figure S5 Plot of Vobs vs. [S] for the oxygenation of p-substituted phenol by acid-treated
tyrosinase Figure S6 Plot of Vobs vs. [S] for the oxygenation of p-substituted phenol by trypsin-treated
tyrosinase Scheme S1 Proposed catalytic mechanism of phenolase and catecholase activity of tyrosinase Scheme S2 Dopachrome Formation Pathway from Tyrosine Scheme S3 Simplified catalytic system of phenolase reaction of melB tyrosinase Scheme S4 Electrophilic aromatic substitution mechanism for the oxygen atom transfer process
from a (µ-η2:η2-peroxo)dicopper(II) species to a phenol substrate
3
Figure S1 Fig. S1 Time courses of the absorption change at 475 nm for the reaction of tyrosinase (0.155 µM) with (a) L-tyrosine (0.1 mM) and with (b) L-DOPA (0.2 mM) in 10 mM Tris-HCl buffer (pH 7.2) at 25 ºC under aerobic conditions. The pro-tyrosinase (open circle) and acid-treated tyrosinase (square)
4
Figure S2 Fig. S2 Michaelis-Menten plot; Vobs vs. the concentration of L-tyrosine in 0.1 M citrate buffer at pH
6.0. [tyrosinase]= 0.059 µM
5
Figure S3 Fig. S3 (a) The gel filtration chromatography of the intact wild type tyrosinase (solid line) at pH 7.0 and acid-treated tyrosinase (dotted line) at pH 3.0. (b) Determination of molecular weight of the acid-treated tyrosinase in glycine-HCl buffer at pH 3.0. Thyroglobulin (669 kDa), apo-ferritin (443 kDa), dimeric alcohol dehydrogenase (150 kDa), monomeric alcohol dehydrogenase (75 kDa), and dimeric carbonic anhydrase (58 kDa) were used as marker proteins for determination of molecular weight (open circle). Closed circle indicated the results of the acid-treated tyrosinase (112 kDa). The calculated molecular weights are shown in the brackets.
6
Figure S4 Fig. S4 HPLC elution diagram of the final reaction mixture of trypsin treated tyrosinase (0.155 mM) and 4-methylphenol (0.33 mM) in the presence of NH2OH (8.0 mM).
0 5 10 15 20 25
OH
Me
OH
Me
OH
O
Me
O
Buffer
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Figure S5 Fig. S5 Plot of vobs vs [S] for the oxygenation of (a) p-F-phenol, (b) p-Cl-phenol, (c) p-COOMe-phenol by acid-treated tyrosinase. Inset; Hanes-woolf plot for the same reaction.
8
Figure S6 Fig. S6 Plot of vobs vs [S] for the oxygenation of (a) p-Me-phenol, (b) p-F-phenol, (c) p-Cl-phenol, (d) p-COOMe-phenol by trypsin-treated tyrosinase. Inset; Hanes-woolf plot for the same reaction.
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Scheme S1 Scheme S1: Proposed Catalytic Cycle of Phenolase and Catechlase Activity of Tyrosinase.
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Scheme S2 Scheme S2: Dopachrome Formation Pathway from Tyrosine.
OH
HOOC NH2
OH
HOOC NH2
O
HOOC NH2
OH O
OHOH
NHHOOC
OO
NHHOOC
Tyrosine DOPA DOPAquinone
DOPAchrome (475 nm)
Tyrosinase Tyrosinase
11
Scheme S3 Scheme S3: Simplified Catalytic System of Phenolase Reaction of melB Tyrosinase.
CuIICuII
His
His
HisHis
His
His
OH
CuIICuII
His
His
HisHis
His
His
O
CuICuI
His
His
HisHis
His
HisO2
O
R
OH
OH
R
OH
Deoxy form
Oxy form Met form
NH2OH
Borate
R
O
O
R
O
O
B
12
Scheme S4 Scheme S4: Electrophilic Aromatic Substitution Mechanism for the Oxygen Atom Transfer Process from a (µ-η2:η2-Peroxo)dicopper(II) Species to a Phenol Substrate.
CuII CuII
His
His
HisHis
His
His
O
O
R
OH
R
-O
CuII CuII
His
His
HisHis
His
His
O
O
H+
rds k
R
O
CuII CuII
His
His
HisHis
His
His
O
O
H
K
CuII CuII
His
His
HisHis
His
His
OH
OO
R
