Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

Article abstract of DOI:10.1016/j.abb.2015.04.007

Abstract Tyrosinase (EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: E_met, E_oxy, and E_deoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The k_cat for OAP is 75 ± 2 s^-1, KMOAP = 1.8 ± 0.2 mM, KMO₂ = 25 ± 4 μM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k₇ 蠑 k₁). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for ^kcat/KMOAP as [O₂] → 0 μM (1.38 ± 0.06) decreasing to 0.83 ± 0.03 as [O₂] → ∞ reflecting a partially rate limiting μ-OH bond cleavage (E_met) and formation (E_oxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91 ± 0.03 μM^-1s^-1 and 2.59E-2 ± 5.31E-4 s^-1. Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.

Full text of DOI:10.1016/j.abb.2015.04.007

Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Mechanistic studies of the tyrosinase-catalyzed oxidative
cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one
Courtney Washington a, Jamere Maxwell a, Joenathan Stevenson a, Gregory Malone a, Edward W. Lowe Jr. b,
Qiang Zhang a, Guangdi Wang a, Neil R. McIntyre a,
⇑
a Division of Mathematical and Physical Sciences, Department of Chemistry, Xavier University of Louisiana, New Orleans, LA 70125, USA
b Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosinase (EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with verte-
brate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme
that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met
and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy
state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding
2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of var-
ious steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mecha-
Received 6 January 2015
and in revised form 20 April 2015
Available online 14 May 2015
Keywords:
Isotope effect
Tyrosinase
2-Aminophenol
Mechanism
Cyclocondensation
Kinetics
nism of this reaction. The kcat for OAP is 75 2 sꢀ1, KM = 1.8 0.2 mM, KMO = 25
4 lM with substrates
OAP
2
binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP
preferentially binds to the oxy form over the met (k7 ꢁ k1). For the met form, His269 and His61 are the
proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation
of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for
k
cat=KOAP as [O2] ? 0
l
M (1.38 0.06) decreasing to 0.83 0.03 as [O2] ? 1 reflecting a partially rate lim-
M
iting
l-OH bond cleavage (Emet) and formation (Eoxy) following protonation in the transition state. The
coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexa-
dione intermediate to APX, forming at a rate of 6.91 0.03
ences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.
l
Mꢀ1sꢀ1 and 2.59Eꢀ2 5.31Eꢀ4 sꢀ1. Differ-
Ó 2015 Elsevier Inc. All rights reserved.
Introduction
larger with 110 more residues present as loops connecting con-
served secondary structural elements. H-subunits of abTy contain
Tyrosinase (E.C. 1.14.18.1) is a member of the type-3 copper
protein family catalyzing the monophenolase and diphenolase
activity on phenolic/anilinic and catechol, 2-aminophenol and
2-aminoaniline substrates, respectively [1–6]. Representative
members of the type-3 copper protein family include: catechol oxi-
dase (CO), hemocyanin (Hc) and tyrosinase (Ty) though members
have diverse functions. Tyrosinase is associated with pigmentation
catalyzing the first step in melanin biosynthesis – a pigment
ubiquitously present in all phyla [6]. The structure of mushroom
(Agaricus bisporus) tyrosinase (abTy) is a tetrameric protein with 2
heavy (H, 392 aa, ꢂ43 kDa) and 2 light (L, 150 aa, ꢂ14 kDa) chains
(2.30 Å resolution, 2Y9W) [7,8]. As a derivative of the ppo3 gene, the
fungal enzyme is structurally homologous to other tyrosinases, only
13 a-helices, 8 mostly short b-strands and many loops, similar to
known type-3 protein structures including: tyrosinase structures
from Streptomyces castaneoglobisporus [9] and Bacillus megaterium
[10,11] as well as Octopus dolfeini Hc [12] and Ipomoea batatas cat-
echol oxidase [13]. Within the H-subunit, the dicopper binding
domain is comprised of four antiparallel helices (
11) positioned perpendicularly with CuA is coordinated by H61
3), H94 ( 4) and H85 (loop connecting 3 to 4) and CuB by
H259, H263 ( 10) and H296 ( 11). The structure of the L-subunit
a3, a4, and a10,
a
(a
a
a
a
a
a
in abTy is best described as a b-trefoil fold with 12 antiparallel
b-strands assembled in a cylindrical barrel of six 2-stranded sheets
[7]. The L-subunit appears to play a structural role in tetramer
formation positioned 25 Å away from the active site as H-subunit
turnover number is unaffected by its absence.
The di-copper domain in tyrosinase is classified by three distinct
geometric and electronic architectures with alternate function
⇑
Corresponding author.
E-mail address: nmcintyr@xula.edu (N.R. McIntyre).
http://dx.doi.org/10.1016/j.abb.2015.04.007
0003-9861/Ó 2015 Elsevier Inc. All rights reserved.

C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
25
toward substrate oxidation: Edeoxy, Emet, and Eoxy in addition to an
2-aminophenols by tyrosinase has been previously studied show-
ing a mechanism similar to catechol compounds [2]. For o-amino-
phenol oxidation, the turnover number is decreased relative to the
o -diphenol substrates though KM values does not appear to be dra-
matically affected by the structural difference [1]. The resulting
o-quinone imine (Q) undergoes a sequential coupling and cycliza-
tion towards the corresponding planar 2-3H-aminophenoxazi-
n-3-one (APX) structure passing through several proposed
intermediates (Scheme 1) [35,40]. Our current study of
2-aminophenol oxidation by abTy was performed to increase the
present body of data contrasting the oxidase mechanism for
2-aminophenol with o-diphenol. The role of the phenylamine moi-
ety was studied using a mixture of steady-state and transient
phase kinetics, sKIEs, global analysis and molecular modeling stud-
ies to characterize multiple aspects of the enzymatic and
non-enzymatic reaction coordinate for 2-aminophenol oxidation.
Therein, we present experimental evidence supporting a distinct
preference for 2-aminophenol oxidation by the Eoxy form of abTy
(over the more stable Emet form) to produce the o-quinone imine
product which passes through a spectroscopically observed tran-
sient phenyliminocyclodione intermediate prior to APX product
formation.
inactive mixed valent copper form [14]. The oxy-state (Eoxy) of
tyrosinase has molecular oxygen bound in a side-on
l
ꢀ
g2 g2
:
geometry linked to the solvent-bridged met-state ( -OH) and the
l
reduced deoxy-state through the reversible exchange of the ligand
as oxygen (Edeoxy) or peroxide (Emet) [15]. The mechanism for
tyrosinase-catalyzed phenol (monophenolase) and o-diphenol
(diphenolase) oxidation has been investigated using steady-state,
transient phase and kinetic isotope effects [16–22]. The monophe-
nolase (monooxygenase) activity is catalyzed by tyrosinase in the
Eoxy form while the o-diphenolase (oxidase) activity results from
both Emet and Eoxy forms of the enzyme [19]. Several tyrosinase
studies demonstrate that the Eoxy form is stable and subsequently
reduced to Emet with the concomitant oxidation of o-diphenol to
the corresponding o-quinone [23–25]. Evaluation of the abTy
o-diphenolase reaction mechanism demonstrate tight O2 binding
in addition to an increased second-order rate constant for the
o-diphenol substrate to preferentially bind with the Emet state over
the Eoxy form of the enzyme [20]. The monophenolase activity of
tyrosinase utilizes an electrophilic aromatic substitution (EAS)
mechanism for ortho-hydroxylation.
Interestingly, tyrosinase and type-3 model complexes lack an
observable kinetic isotope effect (KIE) with ortho-deuterated
monophenol substrates. This suggests that the ortho-deprotona-
tion step in this mechanism is either extremely slow or concerted
with the oxygen insertion step. The observation of a normal KIE
(>1) was revealed upon deuteration of the phenolic hydrogen for
both phenol and o-diphenol substrates [16,17,26,27]. With proton
inventory solvent KIE (sKIE) studies, the mechanism for monophe-
nolase and diphenolase tyrosinase activity support a partially-rate
limiting proton transfer in both Emet and Eoxy forms attributed to
nucleophilic attack by the corresponding phenolate ion to copper
[16,17].
Structure–function relationships illustrate drastically altered
sKIE values based on the para substituent of the substrate. Therein,
the fractionation factors (/) decrease as the observed sKIE for kcat
increase with each mode of abTy-dependent oxidation. For
monophenolase activity, the lowest fractionation factor values
and highest sKIE were observed for substrates with an ionic para
substituent. For diphenolase activity, the trend is less clear cou-
pling higher fractionation factors with lower sKIE values compared
to those reported for the monophenolase reaction. These finding
are consistent with a general base catalytic mechanism important
to deprotonate substrate phenolic hydrogens that contribute to the
stabilization of slightly altered transition state geometries from
either the Eoxy or Emet reaction coordinates [28–34]. The extent of
transition state stabilization through altered protonic interactions
support the presence of a far different microenvironment for the
diphenolase reaction coordinate compared to that observed for
monophenolase activity.
Materials and methods
Reagents
Mushroom tyrosinase (3300 U/mg), 2-aminophenol, L-ascorbic
acid, D2O, mono-, di- and tri-sodium phosphate, DMSO, DMSO-d6
and purchased from Sigma–Aldrich Co. (St. Louis, MO).
HPLC-grade solvents (acetonitrile, methanol, and water) were pur-
chased from Fisher Scientific (Fair Lawn, NJ). Analytical standards
were purchased from Sigma–Aldrich Co. (St. Louis, MO). All other
experimental reagents were purchased from commercial sources
at highest purity grade available and used without additional mod-
ification. Commercial enzyme was purified following the proce-
dure of Duckworth and Coleman [41] or the IMAC method [42].
Protein concentration was determined by the bicinchoninic acid
assay [43] with bovine serum albumin as standard. Protein purity
was assessed by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis.
Oxygen electrode
Reactions at 37.0 0.1 °C were initiated by the addition of
ꢂ0.10–0.50
lM mushroom tyrosinase (4–5 lL) into 2.0 mL of
100 mM sodium phosphate (pH 6.4). Initial velocities were mea-
sured at varying concentrations of 2-aminophenol and oxygen. Ini-
tial rates were measured by following the tyrosinase-dependent
consumption of O2 using a Yellow Springs Instrument Model
5300 oxygen monitor interfaced with a personal computer using
a Dataq Instruments analogue/digital converter (model DI-158RS)
interfaced to Microsoft Excel through the Windaq module, modi-
fied from McIntyre et al. [44]. Using a Maxtec Low flow oxygen
blender, in situ [O2] was changed by varying the percent mixtures
The precedence for 2-aminophenol oxidase activity has been
previously observed in several copper containing enzymes (laccase
[36], tyrosinase [1] and various homologues such as: griF [37], phe-
noxazinone synthase [38]) as well as biomimetic complexes rang-
ing from copper through manganese [39]. The oxidation of
OH
Intermediate 3
Intermediate 2
Product
Tyrosinase Catalyzed
1/2 O2
Intermediate 1
NH2
H2O
H
N
OH
O
NH2
N
NH2
N
NH2
OH
N
NH2
O
NH2
NH
OH
O
OH
O
O
O
λmax = 432 nm
λmax = 380 nm
λmax = 400 nm
λmax = 350 nm
Scheme 1. Reaction stoichiometry for the tyrosinase catalyzed oxidation of 2-aminophenol to o-quinone imine followed by non-enzymatic coupling with 2-aminophenol
passing through several proposed intermediate structures to give 2-amino-3H-phenoxazin-3-one (product). Please note, estimates for the kmax for intermediates were based
on previous work by Barry et al. [35].

26
C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
of oxygen and argon standardized to air-saturated distilled water
at 37.0 0.1 °C. Turnover numbers were normalized to controls
performed at 8.99 mM 2-aminophenol to account for differences
in specific activity between different lots of enzyme. Background
O2 consumption rates were first determined without enzyme and
were subtracted from the rate obtained upon tyrosinase addition.
Initial rates were determined from the consumption of dissolved
oxygen and converted to o-quinone imine production or
2-aminophenol consumption using a 2:1 substrate or product to
oxygen ratio (Eq. (1)).
2-aminophenol concentration range 0.90–8.99 mM under the same
conditions listed above over a phosphate buffer pH range of 5.8–8.5.
The individual parameters of the kcat=KOAP term (kcat and KM) were
M
determined using Eq. (4) and plotted against pH and fit by a linear
regression function.
kcat ꢄ ½OAPꢃ
rate ¼
ð4Þ
KM þ ½OAPꢃ
The ðkcat=KOAPÞ data versus [O2] was fit to the following expo-
D
M
nential curve in Kaleidagraph™.
1 d½O2ꢃ
d½OAPꢃ
d½Qꢃ
2
3
5
!
!
!
vo ¼ ꢀ
¼ ꢀ2
¼ 2
ð1Þ
2
dt
dt
dt
kcat
KOMAP
kcat
kcat
KOMAP
D
D
D
ꢀax½O2
ꢃ
4
¼
ꢀ
e
KOMAP
Initial rate data was then fit to the bi-substrate Michaelis–Men-
ten equation in SigmaPlot 11.2 using the Enzyme Kinetics module
(version 1.3)
observed
½O2ꢃ!0
lM
½O2ꢃ!1
!
kcat
KOMAP
D
þ
½O2ꢃ!1
kcat½OAPꢃ½O2ꢃ
2
rate ¼
ð2Þ
KOI KM þ KOM ½OAPꢃ þ KM ½O2ꢃ þ ½OAPꢃ½O2ꢃ
OAP
OAP
2
ð5Þ
ð6Þ
where kcat is the turnover number (maximal number of OAP and O2
molecules converted to o-quinone imine per tyrosinase active site
where
!
2
Dk1KOD2
kcat
KOMAP
per second), KOMAP and KMO are the Michaelis constants for
D
a
¼
þ
þ 1
k7 þ k9
2-aminophenol (OAP) and molecular oxygen (O2), respectively,
½O2ꢃ!0
l
M
and KOI is the limiting value of KOM when the [OAP] approaches zero.
2
2
Due to the high degree of error for the KOM parameter using Eq. (2), a
replot of k versus [O2] was fit to Eq. (3) to measure the Michaelis
2
app
cat
Oxy-tyrosinase
The addition of
constant for [O2] under pseudo-first order conditions and fit in
Kaleidagraph™.
L-ascorbic acid and hydroxylamine to tyrosinase
assays prevent substrate oxidation and reduce the Emet form of the
enzyme to Eoxy [45]. Suppression of the UV–vis spectroscopic sig-
nal of oxidized 2-aminopehnol was followed using an OLIS refur-
bished Hewlett–Packard 8452 diode array spectrophotometer.
The relative rate of 2-aminophenoxazin-3-one (APX) formation at
app
cat
k
ꢄ ½O2ꢃ
rate ¼
ð3Þ
KOM þ ½O2ꢃ
2
Molecular modeling
432 nm was measured versus
L-ascorbic acid concentrations
intended to perform the in situ reduction of the o-quinone imine
product ranging from 0 to 6 equivalents (Fig. S.1.).
The stability of the Eoxy form of abTy was examined by mixing
the Edeoxy form with an oxygenated buffer in the absence of the
The initial coordinates of the A. bisporus (mushroom) tyrosinase
at 2.30 Å resolution were obtained from the Protein Data Bank
(http://www.rcsb.org/pdb/2Y9X). The structure was prepared for
simulation using the protein preparation module of Schrodinger
Drug Discovery Suite 2011–2. Water molecules were removed
and copper valence was assigned. In the copper binding domains
2-aminophenol substrate. Therein, 80 lM of the reduced enzyme
(Edeoxy) was prepared anaerobically (5 vacuum-argon gas cycles)
with 6-equivalents of hydroxylamine and rapidly mixed with an
oxygen saturated buffer. The signal for Eoxy was followed at
of the active-site, the
l
ꢀ
g2 g2 oxygen bridged structure (Eoxy
: )
e
345 = 18 mMꢀ1cmꢀ1) with a time course collected at
and
g
2-hydroxyl bridged structures (Emet) were created through
345 nm (
2 ms intervals [24,25]. Consumption of the Eoxy signal in the pres-
ence of the 2-aminophenol substrate used 20 M Eoxy was pre-
the manual insertion of the oxygen species followed by minimiza-
tion. The Coulson charges for the ligands were generated and Glide
XP was used to generate the initial poses. The partial atomic
charges utilizing electrostatic potential fitting of the
protein-bound ligand were calculated using Q-site then
re-docked with these updated charges using Glide XP.
l
pared from the reduced enzyme and oxygen gas (vide supra) then
rapidly mixed with a 1 mM 2-aminophenol solution containing
6 M equivalents of
L-ascorbic acid [20].
All rapid-mix studies were performed using
a
Kintek
Mini-Mixer (KinTek Corp., Austin, TX) at 37 0.1 °C with tempera-
ture maintained with a Neslab RTE-110 water circulator. Data was
collected and processed using an apparatus designed in-house with
supplies purchased from Ocean Optics (Ocean Optics, St. Peters-
burg, FL). Specifically, the signal was collected with the Spec-
traSuite interface using a USB 2000 + XR spectrophotometer and
DH-2000 light source linked to the observation cell through Series
74 collimating lenses (200–2000 nm) and solarization resistant
fiber optic cables. All curve fitting and kinetic simulation of this
data was analyzed with Kaleidagraph™ and Kintek Explorer Pro-
fessional (KinTek Corp., Austin, TX).
Solvent kinetic isotope effects
The dependence of D2O on the catalytic efficiency of 2-aminophenol
oxidation as a function of oxygen tension was performed in a
non-competitive fashion following tyrosinase-dependent oxygen con-
sumption. A 10 mM stock of deuterated 2-aminophenol was prepared
with 10% DMSO-d6 in 50 mM sodium phosphate buffer (pH 6.4)
dissolved in D2O, the reaction buffer was 100 mM sodium phosphate
buffer (pH 6.4) dissolved in D2O. Reactions were initiated at
37.0 0.1 °C with 5 lL of enzyme (0.5 nm), six 2-aminophenol concen-
trations from 0.90 to 8.99 mM and five oxygen concentrations ranging
from 65 to 850
l
M were used, respectively. The kcat=KOAP data was
Non-enzymatic formation of 2-aminophenoxazin-3-one
M
determined using standard Michaelis–Menten kinetics (Eq. (4)) and
plotted against oxygen tension fitting the data to Eq. (5). The depen-
100
lM tyrosinase was mixed with 1 mM 2-aminophenol under
dence of pH on the kcat=KOAP term was performed using the
ambient oxygen saturation at 37 0.1 °C.
M

C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
27
The proposed intermediate was observed at 380 nm and the
2-aminophenoxazin-3-one (APX) product was followed at
(pH 6.4). The reaction was incubated at 37 °C for about 6 h. Prior
to HPLC analysis, samples were precipitated by centrifugation at
room temperature, and the solvent was evaporated with a stream
of nitrogen at 37 °C. The residual solution was applied to 6-mL
SUPELCO C18 solid-phase extraction (SPE) columns pretreated with
water and methanol. The columns were washed with HPLC-grade
water followed by elution with methanol. The effluents were again
concentrated by a nitrogen stream at 37 °C to near dryness before
reconstitution with methanol to 1 mL volume for HPLC injection.
Samples were analyzed on a linear ion trap tandem mass spec-
432 nm (e
432 = 23.4 mMꢀ1cmꢀ1). 3-Dimensional analysis of absor-
bance, time and wavelength were exported from SpectraSuite
(Ocean Optics, St. Petersburg, Fl.) into the Specfit module of Kintek
Explorer Professional (KinTek Corp., Austin, TX) where single value
decomposition (SVD) analysis was performed. The spectra was
reconstructed from the SVD fits then subtracted from the experi-
mental data to ensure the SVD analysis was statistically relevant.
Kinetic constants were constrained by the experimental parame-
ters and calculated using FitSpace to measure the values and show
a contour plot.
trometer (LTQ) interfaced with
a Surveyor HPLC system
(Thermo-Fisher Scientific, West Palm Beach, FL). Chromatographic
separations were achieved by using a Synergi 4u Hydro-RP 80A
column (150 ꢄ 2.0 mm, 4
lm particle size; Phenomenex, Torrance,
Kinetic simulation
CA). Samples were eluted from the analytical column with mobile
phase A (water with 0.1% formic acid) and mobile phase B (acetoni-
trile with 0.1% formic acid) at a flow-rate of 0.2 mL/min over
22 min using a linear gradient of 0–100% B for 13 min; holding at
100% B for 5 min before returning to 0% B.
Kintek Explorer Professional (KinTek Corp., Austin, TX) with the
SpecFit module was used for global analysis through direct numer-
ical integration to simulate experimental data [46–48]. The mech-
anism proposed in Scheme 2 was simulated in two different parts:
catalyzed and non-catalyzed reactions. Each respective path was
modeled using the steady- and transient phase data to constrain
the minimal kinetic mechanism for the reaction. For the
tyrosinase-catalyzed oxidation of 2-aminopehnol to the corre-
sponding o-quinone imine, a global analysis approach was utilized.
Therein, mixtures of pre- and steady-state kinetics were used to
evaluate the catalyzed minimal kinetic model (Scheme 2). The
three states of the enzyme (Edeoxy, Emet and Eoxy) were fit as observ-
ables within this model and the experimental data for Eoxy was
added. The experimental Eoxy data was compared to models show-
ing k1 ꢁ k7 and k1 ꢅ k7 to distinguish the relative ability of Emet
and Eoxy to oxidize 2-aminophenol to o-quinone imine, respec-
tively. For the non-enzymatic coupling and cyclization
(non-catalyzed) reaction of the o-quinone imine (Q) to
2-aminophenoxazin-3-one (APX) the time resolved spectra were
recalculated using a single value decomposition (SVD) analysis cal-
culated from a matrix-representation. This was the ideal method
over global analysis because there were far more wavelengths than
species. Using singular values, the statistical relevance of the
reconstructed spectra (relative contribution and weights) was
compared with experimental values and evaluated through resid-
ual analysis. For the non-catalyzed reaction, an irreversible
two-step kinetic model was used for the formation of APX through
a single intermediate. This intermediate represents the accumula-
tion of a transient species associated with early stage coupling
steps in the reaction of Q with OAP required to form APX. FitSpace
was used to calculate a contour plot for rate constants associated
accumulation and decay of this spectroscopically detected
intermediate.
Results
Product characterization
Tandem LC–MS/MS analysis demonstrates two retention times
associated with substrate and product structure (Fig. S.2.). The first
retention time of 3.41 min corresponds to a high relative intensity
peak (M+1) at 110.09 m/z for 2-aminophenol (MW 109.13), tan-
dem MS/MS analysis of this peak gave a fragmentation pattern
with peaks at 93.17, 92.13 m/z corresponding to the hydroxide loss
(C6H6N+ and C6H5N+) and an 82.16 m/z representing phenol rear-
rangement resulting in an M-28 (CO removal) fragment (C5H6N).
The retention time at 11.60 min shows the highest relative inten-
sity peak (M+1) at 213.06 m/z for 2-amino-3H-phenoxazin-3-one
(MW 212.06). Tandem MS/MS analysis of the 213.06 m/z peak
gives two peaks at 185.03 and 186.11 m/z corresponding to loss
of methylamine (–CNH, 27 m/z and –CNH2, 28 m/z).
Steady and transient state kinetics
The steady-state kinetics for the oxidation of 2-aminophenol to
the corresponding o-quinone imine (Q) was fit to the bi-substrate
Michaelis–Menten Eq. (2). Shown in Fig. 1, the kcat is 75 2 sꢀ1
with a KOMAP of 1.8 0.3 mM with a large error on the KMO and KOD
2
2
values. Replotting the rate of 2-aminophenol oxidation as a func-
tion of oxygen concentration under pseudo-first order conditions
provides a KOM value of 25
4 lM (Fig. S.3.). Shown in Fig. S.2.A,
2
the double reciprocal plot of 1/rate versus 1/[2-aminophenol]
intersects on the abscissa while the 1/rate versus 1/[Oxygen] inter-
sects to the left with a positive y-coordinate, represented as
HPLC–MS/MS analysis
y ¼ ½ETotalꢃꢀ1ððk6 ꢀ kcatÞ=ðk5kcatÞÞ = 0.097 s [20]. A replot of the 1/rate
versus 1/[2-aminophenol] slope determined for each oxygen con-
centration (plotted as 1/[O2]) crosses the ordinate at a value
greater than 0 (Fig. S.4.).
Lyophilized samples were prepared from 8.99 mM (18 micro-
moles) of 2-aminophenol oxidized with 750 pmol mushroom
tyrosinase in 100 mM sodium phosphate (200 micromoles) buffer
The binding of O2 to chemically reduced enzyme prepared
anaerobically (Edeoxy) shows the formation of Eoxy observed at
345 nm to be stoichiometric to the initial concentration of the
enzyme, activating approximately 92%. Following equilibration,
k5[O2]
k3
EMET-S
EDEOXY
EOXY
k6
k11
k13
k8 k7[S]
APX
k1[S] k2
Q
I
the rate of decay for Eoxy was observed to be slow, 6.50 ꢄ 10ꢀ4
-
M sꢀ1 (Fig. S.5.). The Eoxy form of tyrosinase was rapidly mixed
k9
l
EMET
EOXY-S
with 2-aminophenol with L-ascorbic acid for the in situ reduction
of o-quinone imine (Q). Under saturating oxygen conditions, the
consumption of the Eoxy signal corresponding to 13 M enzyme
followed a single exponential decay function corresponding to
3.1 5.6Eꢀ2
Mꢀ1 sꢀ1 (Fig. 2).
Scheme 2. Minimal kinetic mechanism of the tyrosinase dependent catalytic
oxidation of 2-aminophenol (S) to o-quinone imine (Q) and its non-enzymatic
cyclo-addition to 2-amino-3H-phenoxazin-3-one (APX) passing through an inter-
mediate (I).
2
l
l

28
C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
[O ] = 58.52 μΜ
2
60
50
40
30
20
10
0
[O ] = 190.03 μΜ
2
[O ] = 244.14 μΜ
2
[O ] = 372.79 μM
2
[O ] = 640.48 μΜ
2
[O ] = 882.09 μΜ
2
[O ] = 905.46 μΜ
2
0
2
4
6
8
10
[2-aminophenol] (mM)
60
50
40
30
20
10
0
[OAP] = 0.90 mM
Fig. 2. Stopped-flow analysis of Eoxy consumption in the presence of 2-aminophe-
nol. Pre-steady state kinetics were measured at 345 nm upon mixing oxy-
tyrosinase (Eoxy) with 1 mM 2-aminophenol with data fit to a single exponential
function.
[OAP] = 1.60 mM
[OAP] = 3.50 mM
[OAP] = 5.80 mM
[OAP] = 7.00 mM
[OAP] = 8.99 mM
Table 1
Derivation and determination of rate constants from for the 2-aminophenol oxidase
activity from steady-state kinetics according to Scheme 2.
kcat ¼ k3k9=ðk3 þ k9Þ
KI;O KMOAP ¼ k3k6k9=½k5k7ðk3 þ k9Þꢃ ¼ kcatKO2 =k7
2
D
KOM ¼ k3k9=½k5ðk3 þ k9Þꢃ ¼ kcat=k5
2
KOMAP ¼ ½k3k9ðk1 þ k7Þꢃ ¼ ½kcatðk1 þ k7Þ=k1k7ꢃ
kcat
¼ k5 ¼ 3:00 ꢆ 0:0048
l
Mꢀ1 sꢀ1
KOMAP
0
200
400
600
800
1000
k1ꢄk7
kcat
¼
KOMAP
k1ꢄk7
[Oxygen] (μM)
If k7 ꢁ k1 the 2nd order rate constant for OAP binding to Emet becomes
kcat
¼ k1 ¼ 0:0421 ꢆ 0:0048
l
Mꢀ1 sꢀ1
KOMAP
Fig. 1. Steady-state kinetics for the tyrosinase dependent oxidation of 2-aminophe-
nol as a function of oxygen tension. Rates were fit with the bi-substrate Michaelis–
Menten equation (Eq. (2)).
KDO
¼
k6
2
k5
k6 ¼ KOD ꢄ k5 ¼ 97 ꢆ 73 sꢀ1
2
The kinetic complexity of the tyrosinase-dependent oxidation
of 2-aminophenol to o-quinone imine (Q) make direct analysis dif-
ficult (see Appendix for distribution equations). Therefore, elemen-
tary rate constants for the minimal kinetic mechanism presented
in Scheme 2 were determined through a mixture of steady-state
and transient phase analysis. Coefficients within the bi-substrate
Michaelis–Menten equation are defined according to Scheme 2.
Kinetic simulation using global fitting analysis of the proposed
minimal kinetic mechanism (Scheme 2) is shown in Tables 1 and
2. Using Emet, Edeoxy, and Eoxy as ‘observables’ to validate our kinetic
model against the experimentally determined rate for Eoxy con-
sumption (k7) to analyze whether 2-aminophenol preferentially
reacts with Emet or Eoxy (Table 2). Rate constants in our model
assumed that product release (Q) was equal to kcat representing
with k3 and k9 while the rate constants k1, k5, k6, k7 were fixed to
the values derived in Table 1 while k2 and k8 were each fixed to
0.1 sꢀ1. The values for k7 were determined through global fitting
Table 2
Steady-state kinetic parameters and rate constants corresponding to the minimal
kinetic mechanism representing the best global fit analysis of steady and pre-steady
state kinetics of 2-aminophenol (S) oxidation to o-quinone imine (Q) and single value
decomposition (SVD) analysis of the cyclization of Q and S to form 2-amino-3H-
phenoxazin-3-one (APX). Please note that k2 and k8 were fixed to 0.1 sꢀ1 and that k11
and k13 were determined using the FitSpace module of Kintek Explorer Professional.
Steady state kinetic parameters
kcat
KM,OAP
75
1.8 0.2
25
2
sꢀ1
mM
KM;O
4
l
l
M
M
2
KD;O
33 24
2
Rate constants
k1
k2
k3
k5
k6
k7
k8
k9
k11
k13
42.1 4.8
0.1
150
3000
97
804 41
0.1
150
mMꢀ1sꢀ1
sꢀ1
sꢀ1
mMꢀ1sꢀ1
sꢀ1
mMꢀ1sꢀ1
sꢀ1
analysis of the mechanism to be 0.804 0.041 l
Mꢀ1sꢀ1. According
sꢀ1
to Fig. 3 (top), k1 ꢅ k7 displayed strong correlation between the
experimental and theoretical values for Eoxy throughout the time
course. Validation of this model was performed by reversing the
values of k1 and k7 (k7 ꢅ k1) and comparing the experimental val-
ues for Eoxy with simulated values where dramatic shift in the the-
oretical Eoxy away the experimental value was observed (Fig. 3,
bottom).
The mechanism for the non-enzymatic coupling of the o-qui-
none imine (Q) product with 2-aminophenol was predicted to pass
through several intermediates leading to the APX product
6910 31
2.54Eꢀ2 5.31Eꢀ4
mMꢀ1sꢀ1
sꢀ1
(Scheme 1). Fig. 4 shows time resolved spectra for a transient peak
at 380 nm (I) followed by peak at 432 nm (APX). SVD
de-convolution of observable species and spectra reconstruction
demonstrate that the residual of the experimental and simulated
a

C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
29
Fig. 4. Time resolved spectra for 2-amino-3H-phenoxazinone (APX) formation from
2-aminophenol and mushroom tyrosinase.
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
Fig. 3. Model validation correlating simulated data representing the distribution of
tyrosinase enzyme states with experimentally derived data for a time course. Each
simulation was calculated used kinetic data fit to the global model with k7 ꢅ k1
(top) or k7 ꢁ k1 (bottom) where the calculated values were switched and compared
directly with Eoxy values determined experimentally.
spectra are well matched (Fig. S.6.). Analysis of rate constants for
the reaction model with global analysis show k11 and k13 to be
6.91 0.03
l
Mꢀ1sꢀ1 and 2.59Eꢀ2 5.31Eꢀ4Eꢀ6 sꢀ1 (Table 2) with
0
200
400
600
800
1000
contour diagram generated though FitSpace showing the data to be
well constrained within the model (Fig. S.7.). By correlating the
[Oxygen] (μM)
absorbance of APX (
intermediate, a
380 = 7.7 mMꢀ1cmꢀ1 was determined and the
e
432 = 23.4 mMꢀ1cmꢀ1) with the observed
Fig. 5. Solvent kinetic isotope effects on the catalytic efficiency (kcat=KOAP) as a
e
M
extracted data was fit to a time course that reflected concentration
(Fig. S.8.).
function of oxygen tension and fit to Eq. (5). The inset displays the dependence of
(kcat=KOAP) with oxygen concentration.
M
The dependence of sKIE on oxygen tension was studied for
k
cat=KOAP (Fig. 5). The results clearly demonstrate that proton trans-
M
and 3.71 Å for the corresponding phenolic hydrogen of catechol
(Fig. 6). For Eoxy the distance of the 2-aminophenol N–H distance
to the peroxide was 2.42 and 2.34 Å for the OH group of catechol
(Fig. 7). For both Emet and Eoxy active site architectures, both sub-
strates share nearly superimposable benzene ring stacking with
the His296 imidazole moiety and hydrogen bonding with Asn260.
fer is increasingly rate limiting to the catalytic efficiency for the
tyrosinase-dependent oxidation of OAP as [O2] ? 0 mM, extrapo-
lating to a value of D2O(k/K) = 1.38 0.06. As [O2] ? 1, the reac-
tion displays an inverse solvent kinetic isotope effect, 0.83 0.03.
Pertinent to the sKIE studies, pH versus kcat=KOAP showed near con-
M
stant value of ꢂ4 mMꢀ1sꢀ1 over a broad range (5.7–8.5) (Fig. S.8.).
Discussion
Molecular modeling
Oxygen binds with high affinity to mushroom tyrosinase [49].
Binding of O2 to abTy is best characterized as a two-electron
exchange stabilization of triplet O2 [50–52]. In the Eoxy form, the
O2 is reversibly activated through a peroxide dicopper(II) complex
that is highly conserved in type-3 proteins, including Hc and Ty.
Docking orientation in the Emet form shows both
2-aminophenol and catechol to interact with the imidazole side
chain of His263 in the CuB binding domain. The distance between
N -His263 and the anilinic hydrogen of 2-aminophenol was 2.26
e

30
C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
Fig. 6. Binding orientation of 2-aminophenol in the active site of the Eoxy form of mushroom tyrosinase. The active site contains copper coordinating histidines (tube), a
bridging peroxide for CuA and CuB which hydrogen bonds the amino moiety of 2-aminophenol(OAP). For clarity, catechol was not included but (like Emet) shares an
indistinguishable binding mode with 2-aminophenol. All other residues that extend beyond the active site are represented by their secondary structural elements.
Fig. 7. Homologous binding mode of 2-aminophenol and catechol (‘Substrates’) in the active site of the Emet form of mushroom tyrosinase. Please note, the Emet active site
contains copper coordinating histidines (tube) with bridging hydroxide (ball and stick) ion for CuA and CuB. Please note the hydrogen bonding between the hydroxyl moiety
of each substrate with Asn260. All other residues that extend beyond the active side are represented by their secondary structural elements.
Binding of O2 to the dicuprous Edeoxy state results in filling the p⁄
HOMO (highest occupied molecular orbital) through two electron
reduction to peroxide (Oꢀ2 2) to overcome the spin-forbidden triplet
state oxygen (3O2) containing two unpaired electrons situated in
the doubly degenerate p⁄ orbital. In the Eoxy form, the side-on
the difference in KOM and KDO values is relevant (Figs. S.3. and
2
2
S.4.) [55]. With KOM < KDO , there is synergistic binding of substrates
suggesting 2-aminophenol binds tighter to the enzyme in the pres-
ence of oxygen to form Eoxy. The positive y-coordinate within the
x,y intersection in the 1/rate versus 1/[oxygen] (Fig. S.2.A) indicates
2
2
l
ꢀ
g2 2 geometry is responsible for direct orbital overlap (coor-
:g
that the rate of O2 release is faster than that of Q release (k6 > kcat
)
dination) necessary to produce the intense ligand to metal charge
transfer (LMCT) band and is the most favorable orbital overlap for
dioxygen reduction to peroxide (Eoxy) [53]. The resulting peroxide
which is further demonstrated through derivation of this rate con-
stant in the Table 1 (k6 = 97 and kcat = 75 sꢀ1). Therefore, the bind-
ing regime for substrates to the Edeoxy form is best described as a
steady-state ordered mechanism with high error in the KOD mea-
surement attributed to the tighter binding of the oxygen to the
type-3 catalytic center compared with 2-aminophenol [49]. Tran-
sient state kinetics following the Eoxy form of abTy show the signal
for the normally stable Eoxy active site architecture to be consumed
in single exponential fashion in the presence of 2-aminophenol
(Figs. S.5. and 2). The rate for this process (k7) was determined to
be over 15-fold faster than the corresponding rate for the Emet
character of the LMCT is associated with Oꢀ2 2 pr⁄ ? Cu(II) (dx2ꢀy2
)
2
interactions responsible for significant peroxide character in the
LUMO. These p⁄r interactions allow strong
r
donation into dx2
of
ꢀy2
Cu allowing a p⁄r superexchange pathway to stabilize the singlet
state of the Cu2/O2 complex and subsequently weaken the O–O bond
resembling a bis-l-oxo complex [54].
The oxidation of 2-aminophenol to o-quinone imine (Q) by abTy
was explored using steady-state kinetic analysis (Fig. 1). The sim-
ilarity in KOM and KOD values (25 4 and 33 24
lM) using the
2
2
(k1) form of abTy derived from kcat=KOAP (Table 2). The observation
that k7 ꢁ k1 supports a model of preferred 2-aminophenol binding
to Eoxy over Emet which is in stark contrast to work pursued with
o-diphenol substrates [20]. The rate for k7 became an experimental
M
bi-substrate Michaelis–Menten Eq. (2) suggests an equilibrium
ordered mechanism for substrate binding, though the replot of
the slopes in the 1/rate versus 1/[OAP] plot versus 1/[O2] clearly
show the line does not pass through the origin, supporting that

C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
31
(A)
(C)
(B)
HO
NH2
(N)His29 6
His6 1(N)
H
O
HN
H+
(N)His296
O
HN
H
+(N)His29 6
His6 1(N)
(N)His61
His85 (N)
His9 4(N)
CuB(II)
(N)His259
(N)His2 63
His8 5(N)
His94 (N )
CuB(II)
(N)His2 59
(N)His26 3
CuA(II)
CuA(II)
(N)His2 59
(N)His26 3
His8 5(N)
His94 (N )
CuA
(I)
CuB(I)
O
H
O
H
H2O
me t form
H2O
+
H2O
+
NH
O
NH
O
(F)
(E)
H
O
NH
HO
NH2
(D)
O2
H
+H
(N)His296
+ H
(N)His29 6
+H
(N)His296
(N)His259
His6 1(N)
His6 1(N)
His8 5(N)
His6 1(N)
His8 5(N)
O
O
(N)His259
(N)His2 59
(N)His26 3
His85 (N)
CuA(II)
CuB(II)
CuA(II)
CuB(II)
CuA(I)
CuB(I)
(N)His2 63
His9 4(N)
His94 (N )
His94 (N )
(N)His2 63
O
O
oxy form
reduced form
Scheme 3. Proposed mechanism for the tyrosinase dependent oxidation of 2-aminophenol to o-quinone imine.
constraint for the global fitting analysis of the rate constants to val-
idate our proposed model of 2-aminophenol oxidation where
k7 ꢁ k1 (Table 2). Using global analysis, the best correlation of
the experimentally determined values for k7 was observed for
the k7 ꢁ k1 simulation supporting our model that 2-aminophenol
preferentially binds to the Eoxy rather than Emet form of abTy
(Fig. 3).
formation and consumption in the Emet state. Relative to the transi-
tion from Eoxy to Emet, the extent of l-OH bond formation is repre-
sented as the inverse sKIE (D(kcat=KOAP) < 1) region of the curve at
M
high [O2]. The increase toward the normal sKIE values
(D(kcat=KOAP) > 1 as [O2] decreases) demonstrate the partial rate
M
limitation of l-OH bond cleavage as Emet is converted to Edeoxy fol-
lowing protonation in the transition state. Compared with
2-aminophenol oxidase sKIE values, the EAS mechanism for phenol
hydroxylation by Eoxy display the highest sKIE values and support
an altered binding mode compared with o-diphenol substrates
[56–58]. Therefore, the transition from Eoxy to Emet is
rate-limiting for phenol hydroxylation while it serves as a thermo-
dynamic drive for the 2-aminophenol oxidase reaction.
An independent measure of the increased reactivity of the
o-aminophenol substrates for Eoxy over Emet was further investi-
gated using solvent KIE (sKIE). Previous sKIE values measured at
ambient oxygen concentrations show normal sKIE values and an
accompanying decrease in fractionation factors from reactant to
product state (/R ꢂ 1 ? /P < 1) for both mono- and di-phenolase
activity that results from a single protonic interaction (n = 1)
[16,17]. The behavior of the same exchangeable position in the
abTy reaction coordinate reveal distinct differences from the reac-
tant and product (and presumably the transition) states distinct to
the reactions of both Emet and Eoxy states. Our present sKIE study
sought to identify the distribution and relative rate limitation of
bridging protonic interactions within the transition state struc-
tures for both Emet and Eoxy forms of the enzyme. To study this
effect relative to the abTy enzyme forms present in 2–aminophenol
oxidation (Emet and Eoxy), oxygen tension was varied with data rep-
Based on sKIE results from this study and others, a mechanism
was postulated to address the unexpected preference of
2-aminophenol for Eoxy form of abTy. In Scheme 3, pose (A) has
the 2-aminophenol substrate orienting itself with the anilinic
group near His296. Structure (C) follows anilinic group
de-protonation by His296. Based on crystal structure data for abTy
and B. megaterium tyrosinase there is precedence for numerous
stable conformations and copper mobility present in type-3 dicop-
per architectures [7,9,59]. Therein, multiple metal ion binding
modes for this structure were described through a cooperative
model describing high copper ion plasticity controlling reactivity
and substrate selectivity of the active site coppers is thought to
limit CO to diphenolase activity and Hc to reversible oxygen bind-
ing. In support of copper ion plasticity and sampling between dif-
ferent enzyme forms, the reversible oxygen binding with Hc was
altered through the addition of a denaturant (SDS) that moved
blocking residues opening the active site to the catalytic oxidation
of the diphenolic substrate dopamine while CO has copper coordi-
nating histidine residues positioned in a conformationally labile
loop regions [60,61]. Based on our molecular modeling studies,
the proposed binding orientations for 2-aminophenol and catechol
are essentially superimposable with respect to benzene ring posi-
tioning near H296 and proximity to the CuA–CuB binding domains
for both Emet and Eoxy forms of abTy (Figs. 6 and 7). The similarity in
resented as
k
cat=KOAP (Fig. 5). We chose catalytic efficiency
M
(kcat=KOAP) over kcat or KM based on the derivations present in
M
Table 1. Specifically, this term involves both the binding and chem-
ical steps of 2-aminophenol oxidation and is well suited to discrim-
inate proton transfer events between with Emet and Eoxy forms of
abTy. This experimental design requires that the pH-rate profile
be independent of the catalytic efficiency. The constant pH-rate
profile demonstrates that kinetic data collected in either protiated
or deuterated solvent are isolated to the di-coppers involved in
catalysis and cannot be attributed to matrix perturbations associ-
ated with pH versus pD effects (Fig. S.9.). Our results show a
dramatic change in the magnitude of the sKIE as a function of
oxygen concentration supporting a model in which the magnitude
of the sKIE values are a direct transition state probe of
l-OH

32
C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
spatial orientation providing a
p
–
p
binding interaction with
Following release of the o-quinone imine product, tandem cou-
pling and cyclization of o-quinone imine (Q) with 2-aminophenol
(OAP) to forms the isolated product 2-amino-3H-phenoxazin-3-
one (APX) (Figs. S.2. and 4). Previous work on melanin carried out
by Riley [62] and dopamine o-quinone cyclization [63] explored
intramolecular cyclization for compounds biologically relevant to
melanogenesis. Based on proposed intermediates and projected
kmax values, the spectroscopic signals observed at 380 and 432 nm
corresponding to intermediate 2 and product in the nucleophilic
intermolecular conjugate addition pathway for APX formation
(Scheme 1). Using SVD, the rate of coupling is much faster than the
His296 and the benzene ring proposes that any difference in reac-
tivity is solely due to differences in the functional group substitu-
tion of aniline for a phenolic moiety within the o-diphenol
substrate scaffold.
Beginning with the Emet form of the enzyme, we propose a
mechanism in which the l-phenylamine of 2-aminophenol coordi-
nates the dicopper domain resulting in a conformational change in
the tertiary structure. Similar to reversible O2 binding in the type-3
proteins, the
priate superexchange pathway while increasing the hydroxide ion
character of the solvent derived -OH allowing the capture of a
l-phenylamine structure would maintain the appro-
l
corresponding cyclization (6.91 0.3 l
Mꢀ1sꢀ1 and 2.59
phenolic proton by His61 allowing reduction of the copper
domains to Edeoxy with concomitant oxidation to the o-quinone
imine (Q) product (Complex C). The resulting Edeoxy form binds
molecular oxygen then 2-aminophenol where the bases in this
5.31Eꢀ4 sꢀ1) passing through a phenyliminocyclohexadione inter-
mediate(Table 2). Using the sequentialmodelproposedin Scheme1,
the extracted data suggest the short lived o-quinone imine (Q) enzy-
matic product undergoes
a
Michael-type addition with
form are the
l
ꢀ
g2
:
g2 dicopper peroxide for the anilinic roton
2-aminophenol resulting in an observed phenyliminocyclohexa-
dione intermediate that cyclizes to the APX product (Fig. 4).
and His61 for the phenolic proton followed by coordination to
the copper centers. The resulting 2eꢀ, 2H+ redox process yields
the second o-quinone imine and water with His61 deprotonated
In conclusion, this study shows the oxidative cyclocondensation
of 2-aminohenol to undergo an abTy dependent oxidation to its
corresponding o-quinone imine releasing a water molecule from
both Emet and Eoxy followed by several conjugate addition interme-
diates leading to the eventual 2-amino-3H-phenoxazin-3-one
(APX) product (Scheme 3). Within this reaction, the
abTy-dependent oxidation of 2-aminophenol was preferentially
carried out by the Eoxy rather than Emet forms of the enzyme. We
suggest the additional stabilization of the Emet form by the anilinic
group of 2-aminophenol relative to o-diphenol substrates as an
explanation for the difference in reactivity (Scheme 3). The sKIE
studies support this model as the rate limitation for proton transfer
is greatest with the Emet form and lowest (inverse) with Eoxy. This
to give the characteristic l-OH of Emet (Complexes E and F).
From a combination of kinetic and molecular modeling coupled
with solvent kinetic isotope effects, it appears that the Emet form
has a greater degree of conformational sampling allowing stable
quasi-productive binding modes to occur. The
(complex B) is one postulated intermediate that could cause the
reduction of the -OH of Emet to become slow or rate-limiting to
l-phenylamine
l
the global catalytic model (Scheme 2). Additional support for con-
formational mobility in the abTy active site is found by correlating
secondary structural elements of the protein with ligand and bind-
ing pocket surfaces (Figs. 8 and 9). With respect to 2-aminophenol
binding modes in both Emet and Eoxy, the anilinic moiety is buried
in the surface accessible active-site. With a similar interaction with
His296 for each enzyme form, the resulting loop extending from
suggests that l-OH formation (Eoxy to Emet) is favorable compared
the rate limiting transition of Emet to Edeoxy likely caused by the for-
mation of a quasi-stable conformation of the Emet containing a
the
a
11 helix defines a significant portion of the substrate binding
l-phenylamine bridge between CuA and CuB that mimics the
face. Following proton transfer, subsequent electronic changes in
the active site would be expected to sufficiently affect the loop
conformation allowing the phenolic group to position near the
CuA domain for deprotonation with H61.
bis- -oxo dicopper geometry observed in the Eoxy form of abTy.
l
Overall, the present study characterized distinct mechanistic dif-
ferences in the conventional mechanism between o-diphenolase
and 2-aminophenol oxidase with abTy and followed the
Fig. 8. Representation of the ligand and active site binding surface for 2-aminophenol and the Emet form of mushroom tyrosinase. Please note, 2-aminophenol is shown in
blue, the binding pocket is covered with a gray with the loop (teal) extending from the
reader is referred to the web version of this article.)
a11-helix (green). (For interpretation of the references to color in this figure legend, the

C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
33
Fig. 9. Binding orientation of 2-aminophenol in the CuB domain of Eoxy form of mushroom tyrosinase. Please note, 2-aminophenol is shown in blue, the binding pocket is
covered with a gray showing the loop (teal) extending from the
the web version of this article.)
a11-helix (green). (For interpretation of the references to color in this figure legend, the reader is referred to
Eoxy
ET
non-enzymatic cyclization through a previously undetected inter-
mediate leading to the APX product.
¼ k1k3k5k8½O2ꢃ½Sꢃ þ k3k5k8½O2ꢃ þ k2k5k8½O2ꢃ þ k2k8
þ k1k3k5k9½O2ꢃ½Sꢃ=
D
Acknowledgments
Eoxy ꢀ S
2
¼ k1k3k5k7½O2ꢃ½Sꢃ þ k3k5k7½O2ꢃ½Sꢃ þ k2k5k7½O2ꢃ½Sꢃ þ k2k7½Sꢃ
ET
Neil R. McIntyre would like to acknowledge support from
NIH-1SC3GM112558-01, NIH-RCMI Grant 2G12MD007595-06,
Louisiana Cancer Research Consortium (LCRC), NSF-Louis Stokes
Alliance for Minority Participation (JM), Center for Undergraduate
Research (CUR)(JS), Louisiana Board of Regents Research Commer-
cialization and Education Enhancement Program (RCEEP)(GM),
Department of Chemistry Work/Study. Edward W. Lowe, Jr.
acknowledges NSF support through the CI-TraCS fellowship
(OCI-1122919). Guangdi Wang would like to acknowledge support
from Department of Defense grant W81XWH-04-1-0557, Office of
Naval Research Grant N00014-99-1-0763, and LCRC. We would
also like to acknowledge the insightful recommendations of Dr.
Larry Byers (Tulane) during the preparation of this manuscript.
þ k2k6=
D
Please note the ET is total enzyme and represents the sum of all
distribution equations for each enzyme form.
Rate of product formation:
dQ
¼ k3½Emet ꢀ Sꢃ þ k9½Eoxy ꢀ Sꢃ
dt
2
dQ=dt ¼ ðk3k1k6k8½Sꢃ þ k6k8 þ k1k5k7k9½O2ꢃ½Sꢃ þ k1k7k9½O2ꢃ½Sꢃ
2
þ k1k6k9½Sꢃ þ k9k1k3k5k7½O2ꢃ½Sꢃ þ k3k5k7½O2ꢃ½Sꢃ
þ k2k5k7½O2ꢃ½Sꢃ þ k2k7½Sꢃ þ k2k6Þ=ðk2k6k8 þ k3k5k7k9½O2ꢃ½Sꢃ
þ k2k5k7k9½O2ꢃ½Sꢃ þ k2k7k9½Sꢃ þ k2k6k9 þ k1k6k8½Sꢃ þ k6k8
þ k1k5k7k9½O2ꢃ½Sꢃ þ k1k7k9½O2ꢃ½Sꢃ þ k1k6k9½Sꢃ
þ k1k3k6k8½Sꢃ þ k3k6k8 þ k2k6k8 þ k1k3k7k9½Sꢃ
2
Appendix A
2
Distribution Equations
þ k1k3k6k9½Sꢃ þ k1k3k5k8½O2ꢃ½Sꢃ þ k3k5k8½O2ꢃ þ k2k5k8½O2ꢃ
þ k2k8 þ k1k3k5k9½O2ꢃ½Sꢃ þ k1k3k5k7½O2ꢃ½Sꢃ þ k3k5k7½O2ꢃ½Sꢃ
Emet
ET
2
¼ k2k6k8 þ k3k5k7k9½O2ꢃ½Sꢃ þ k2k5k7k9½O2ꢃ½Sꢃ þ k2k7k9½Sꢃ
þ k2k5k7½O2ꢃ½Sꢃ þ k2k7½Sꢃ þ k2k6Þ
þ k2k6k9=
D
If k3 ꢁ k2 and k9 ꢁ k8
2
2
dQ
dt
k1k5k7k9½O2ꢃ½Sꢃ þ k1k7k9½O2ꢃ½Sꢃ þk1k6k9½Sꢃ þ k9k1k3k5k7½O2ꢃ½Sꢃ þk3k5k7½O2ꢃ½Sꢃ
¼
2
2
2
k3k5k7k9½O2ꢃ½Sꢃ þ k1k5k7k9½O2ꢃ½Sꢃ þk1k7k9½O2ꢃ½Sꢃ þ k1k6k9½Sꢃ þ k1k3k7k9½Sꢃ þ k1k3k6k9½Sꢃ þ k1k3k5k9½O2ꢃ½Sꢃ þ k1k3k5k7½O2ꢃ½Sꢃ þ k3k5k7½O2ꢃ½Sꢃ
Emet ꢀ S
2
Appendix B. Supplementary data
¼ k1k6k8½Sꢃ þ k6k8 þ k1k5k7k9½O2ꢃ½Sꢃ þ k1k7k9½O2ꢃ½Sꢃ
ET
þ k1k6k9½Sꢃ=
D
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.abb.
2015.04.007.
Edeoxy
ET
2
¼ k1k3k6k8½Sꢃþk3k6k8 þk2k6k8 þk1k3k7k9½Sꢃ þk1k3k6k9½Sꢃ=
D

34
C. Washington et al. / Archives of Biochemistry and Biophysics 577–578 (2015) 24–34
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