Inhibition of tyrosine phenol-lyase by tyrosine homologues

Article abstract of DOI:10.1007/s00726-016-2263-7

We have designed, synthesized, and evaluated tyrosine homologues and their O-methyl derivatives as potential inhibitors for tyrosine phenol lyase (TPL, E.C. 4.1.99.2). Recently, we reported that homologues of tryptophan are potent inhibitors of tryptophan indole-lyase (tryptophanase, TIL, E.C. 4.1.99.1), with K_i values in the low μM range (Do et al. Arch Biochem Biophys 560:20–26, 2014). As the structure and mechanism for TPL is very similar to that of TIL, we postulated that tyrosine homologues could also be potent inhibitors of TPL. However, we have found that homotyrosine, bishomotyrosine, and their corresponding O-methyl derivatives are competitive inhibitors of TPL, which exhibit K_i values in the range of 0.8–1.5?mM. Thus, these compounds are not potent inhibitors, but instead bind with affinities similar to common amino acids, such as phenylalanine or methionine. Pre-steady-state kinetic data were very similar for all compounds tested and demonstrated the formation of an equilibrating mixture of aldimine and quinonoid intermediates upon binding. Interestingly, we also observed a blue-shift for the absorbance peak of external aldimine complexes of all tyrosine homologues, suggesting possible strain at the active site due to accommodating the elongated side chains.

Full text of DOI:10.1007/s00726-016-2263-7

Amino Acids
DOI 10.1007/s00726-016-2263-7
ORIGINAL ARTICLE
Inhibition of tyrosine phenol‑lyase by tyrosine homologues
Quang Do1,3 · Giang T. Nguyen1,4 · Robert S. Phillips1,2
Received: 26 December 2015 / Accepted: 20 May 2016
© Springer-Verlag Wien 2016
Abstract We have designed, synthesized, and evaluated
tyrosine homologues and their O-methyl derivatives as
potential inhibitors for tyrosine phenol lyase (TPL, E.C.
4.1.99.2). Recently, we reported that homologues of tryp-
tophan are potent inhibitors of tryptophan indole-lyase
(tryptophanase, TIL, E.C. 4.1.99.1), with Ki values in the
low µM range (Do et al. Arch Biochem Biophys 560:20–
26, 2014). As the structure and mechanism for TPL is very
similar to that of TIL, we postulated that tyrosine homo-
logues could also be potent inhibitors of TPL. However,
we have found that homotyrosine, bishomotyrosine, and
their corresponding O-methyl derivatives are competitive
inhibitors of TPL, which exhibit Ki values in the range of
0.8–1.5 mM. Thus, these compounds are not potent inhibi-
tors, but instead bind with affinities similar to common
amino acids, such as phenylalanine or methionine. Pre-
steady-state kinetic data were very similar for all com-
pounds tested and demonstrated the formation of an equili-
brating mixture of aldimine and quinonoid intermediates
upon binding. Interestingly, we also observed a blue-shift
for the absorbance peak of external aldimine complexes
of all tyrosine homologues, suggesting possible strain at
the active site due to accommodating the elongated side
chains.
Keywords Pyridoxal-5′-phosphate · Reaction mechanism ·
β-Elimination · Stopped-flow kinetics · Amino acid
homologue
Abbreviations
PLP
TPL
TIL
Pyridoxal-5′-phosphate
Tyrosine phenol-lyase [EC 4.1.99.2]
Tryptophan indole-lyase [EC 4.1.99.1]
SOPC S-(o-Nitrophenyl)-l-cysteine
Introduction
Tyrosine phenol-lyase (TPL, [E.C. 4.1.99.2]) is a pyridoxal-
5′-phosphate (PLP)-dependent bacterial enzyme that cata-
lyzes the reversible hydrolytic cleavage of the Cβ–Cγ bond
of l-Tyr to phenol and ammonium pyruvate (Eq. 1) (Yam-
ada et al. 1968; Kumagai et al. 1970a, b). In addition to
its physiological substrate, TPL also catalyzes the in vitro
β-elimination of substrates with good leaving groups on the
β-carbon, including l-serine, l-cysteine, S-(o-nitrophenyl)-
l-cysteine (SOPC) (Eq. 2), O-acetyl-l-serines, and S-alkyl-
l-cysteines (Eq. 3) (Yamada et al. 1968; Kumagai et al.
1970a; Phillips 1987). As the cleavage of l-Tyr is revers-
ible, TPL was also utilized in the enzymatic production of
l-Tyr (Phillips et al. 1989) and β-substitution reactions in
the synthesis of 3,4-dihydroxyphenyl-l-alanine (L-dopa)
(Kumagai et al. 1969), and aza-l-tyrosine (Watkins and
Phillips 2001).
Handling Editor: C. Schiene-Fischer.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-016-2263-7) contains supplementary
material, which is available to authorized users.
* Robert S. Phillips
plp@uga.edu
1
Department of Chemistry, University of Georgia, Athens, GA
30602, USA
2
Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, GA 30602, USA
3
Present Address: Hill Manufacturing Co., Inc., 1500
Jonesboro Rd SE, Atlanta, GA 30315, USA
4
Present Address: Adelaide, Australia
1 3

Q. Do et al.
(1)
(2)
(3)
The postulated mechanism of TPL consists of sev-
eral steps, in which formation of a quinonoid carbanionic
intermediate is an essential step of the catalytic cycle
(Scheme 1) (Muro et al. 1978; Phillips et al. 2002, 2006;
Milić et al. 2011). The first step is transaldimination, in
which the internal aldimine is transformed into an exter-
nal aldimine upon binding of the substrate. Subsequent
deprotonation of the Cα proton, by the amino side chain of
Lys257, gives rise to the quinonoid intermediate. Ketoni-
zation of the phenol, accompanied by Cγ protonation,
subsequently leads to the cleavage of the Cβ–Cγ bond to
form free phenol and an aminoacrylate intermediate. Pre-
viously, we and others studied kinetics and structures of
quinonoid complexes and provided further evidence for
a quinonoid structure as the key intermediate in the TPL
mechanism (Muro et al. 1978; Phillips et al. 2002, 2006;
Scheme 1 Proposed β-elimination mechanism of TPL
1 3

Inhibition of tyrosine phenol-lyase by tyrosine homologues
Milić et al. 2008, 2011). Furthermore, the crystal structure
of F448H TPL with a bound substrate shows evidence for
a contribution of ground state strain to catalysis, since a
closed conformation of the quinonoid intermediate shows
the substrate aromatic ring bent ~20° out of plane (Milić
et al. 2011). However, it remains unclear if Cγ proton trans-
fer and Cβ–Cγ cleavage follow a stepwise or concerted
mechanism.
Tryptophan indole-lyase (tryptophanase, TIL, E.C.
4.1.99.1) is another PLP-dependent bacterial enzyme that
catalyzes the analogous reversible (Watanabe and Snell
1972) hydrolytic cleavage of the Cβ–Cγ bond of trypto-
phan to indole and ammonium pyruvate (Snell 1975). TPL
and TIL from various bacteria share about 40 % sequence
identity, and both enzymes adopt virtually identical three-
dimensional folds (Antson et al. 1993; Isupov et al. 1998).
Also, the accepted mechanism for TIL (Phillips 1991,
2000; Do et al. 2014) is remarkably similar to that sug-
gested for TPL in the cleavage of their respective sub-
strates. However, the two enzymes show strict specificity
for their physiological substrates. Recently, we reported the
evaluation of mechanism-based inhibitors for TIL and dis-
covered that homologation of the physiological substrate,
l-tryptophan, resulted in potent inhibitors of TIL (Do et al.
2014). Our rationale was to design a transition-state analog
inhibitor resembling the elongated transition-state structure
of the indolenine intermediate, or a bi-product analog of
the phenol-aminoacrylate complex immediately after bond
cleavage. Since TIL and TPL have similar structures and
mechanisms, and with the encouraging results from our
previous work, we were interested in extending our design
and rationale to search for new mechanism-based inhibitors
of TPL. Inhbitors of TIL have played a role in the determi-
nation of the physiological effects of the indole product (Di
Martino et al. 2002; Venkatesh et al. 2014), and the avail-
ability of comparable potent TPL inhibitors may assist in
elucidation of the hitherto unknown physiological function
of the phenol produced by TPL.
Fig. 1 Structures of L-tyrosine and L-tyrosine homologues and
derivatives
O-methyl-l-homotyrosine (6a) and O-methyl-l-bishomo-
tyrosine (6b), as it would be of interest to explore whether
methylation of the phenol OH would have an effect on the
efficiency of the proposed compounds as inhibitors for
TPL.
Materials and methods
Materials: Methylene chloride and ethanol were previ-
ously dried over CaH2 and Mg, respectively, prior to use.
2-(4-Methoxyphenyl)ethanol (Acros), 3-(4-methoxyphe-
nyl)propan-1-ol (Aldrich), triphenylphosphine (Aldrich),
carbon tetrabromide (Janssen Chimica), diethyl acetamid-
omalonate (Aldrich), Aspergillus acylase I (0.43 unit/mg,
Aldrich), pyridoxal-5′-phosphate (PLP) (USB Corp.), and
all other reagents (Thermo Fisher Scientific) were used
without further purification. S-(o-Nitrophenyl)-l-cysteine
(SOPC) used in enzyme assays was prepared as previously
described (Phillips et al. 1989). Enzyme assays were per-
formed using distilled deionized water. All NMR data were
collected on a 400-MHz Varian Mercury Plus NMR instru-
ment, and data were processed by Mestrec MNova NMR
processing software. ESI–MS experiments were performed
on a Perkin Elmer Sciex API I Plus.
In the present work, we postulated that inhibitors that
structurally resemble the elongated transition-state struc-
ture leading from the proposed keto-quinonoid interme-
diate to the product complex can potentially inhibit TPL.
Based on that rationale, we proposed that homologues of
the physiological substrate, l-tyrosine, may inhibit TPL
activity. This work describes the synthesis, steady-state,
and pre-steady-state kinetic evaluations of L-homotyrosine
(1a) and L-bishomotyrosine (1b) as possible mechanism-
based inhibitors for TPL (Fig. 1). The quinonoid interme-
diate was also proposed to form a closed conformation in
which hydrogen bonding from the hydroxyl group con-
tributes to the Cα–Cβ–Cγ angle strain to assist in the cleav-
age of the Cβ–Cγ bond (Milić et al. 2011). Hence, we also
evaluated methylated derivatives of tyrosine homologues,
Synthesis of L‑homotyrosine, L‑bishomotyrosine,
and O‑methyl derivatives
1-(2-Bromoethyl)-4-methoxybenzene (3a): In a three-neck
flask, 2-(4-methoxyphenyl)ethanol (2a, 3.00 g, 1 eq.) and
triphenylphosphine (6.72 g, 1.3 eq.) were dissolved in dry
CH2Cl2 (25 ml). In an addition funnel, carbon tetrabromide
(8.50 g, 1.3 eq.) was dissolved in dry CH2Cl2 (15 ml) and
added dropwise under inert atmosphere at 0 °C until the
addition was complete. The reaction was allowed to stir
at room temperature for an additional 4 h, or until com-
plete disappearance of starting materials from thin-layer
chromatography (TLC). The solvent was removed under
reduced pressure, and the residue was purified by column
1 3

Q. Do et al.
chromatography using hexane and EtOAc to yield 3a as a
clear oil. Yield: 4.11 g (97 %). 1H NMR (CDCl3) δ (ppm):
3.08–3.12 (t, 2H), 3.51–3.55 (t, 2H), 3.79 (s, 3H), 6.85–
6.87 (d, 2H), 7.12–7.14 (d, 2H).
cooled, filtered, and washed with water to give 1a as a
white solid. Yield: 68 mg (73 %). 1H NMR (D2O/NaOD) δ
(ppm): 1.54–1.62 (m, 2H), 2.24–2.28 (t, 2H), 3.04 (s, 1H),
6.34–6.36 (d, 2H), 6.79–6.81 (d, 2H).
Diethyl 2-acetamido-2-(4-methoxyphenylethyl)malonate
(4a): In a three-neck flask, diethyl acetamidomalonate
(5.25 g, 1.3 eq.) was added to a solution of dry etha-
nol (40 ml) containing dissolved sodium metal (0.556 g,
1.3 eq.) at 0 °C. The mixture was stirred for an additional
30 min at 0 °C. Then, compound 3a (4.00 g, 1 eq.) was
added in, and the solution was allowed to reflux under
inert atmosphere for an additional 15 h. The solvent was
removed under reduced pressure, and the residue was puri-
fied by column chromatography using hexane and EtOAc.
Subsequent removal of the eluting solvent under reduced
pressure yielded compound 4a. Yield: 3.30 g (51 %).1H
NMR (CDCl3) δ (ppm): 1.23–1.27 (t, 6H), 2.00 (s, 3H),
2.40–2.43 (m, 2H), 2.64–2.68 (m, 2H), 3.78 (s, 3H), 4.19–
4.22 (m, 4H), 6.77 (s, 1H), 6.80–6.82 (d, 2H), 7.05–7.07 (d,
2H).
2-Acetamido-4-(4-methoxyphenyl)butanoic acid (5a): In
a round-bottom flask, NaOH (0.717 g, 2 eq.) was dissolved
in aqueous tetrahydrofuran (1:1, THF:H2O, 40 ml) solution,
then compound 4a (3.15 g, 1 eq.) was added, and the solu-
tion was allowed to reflux for 15 h. The solvent was then
removed under reduced pressure, and the resulting residue
was taken up in water and EtOAc. The aqueous layer was
acidified to pH 2 using 6-M HCl and extracted with EtOAc.
The organic layer was washed with water and dried over
MgSO4. Removal of the solvent under reduced pressure
gave 5a as a white solid. Yield: 2.17 g (96 %). 1H NMR
(DMSO) δ (ppm): 1.94 (s, 3H), 3.78 (s, 3H), 4.12–4.15 (m,
1H), 6.90–6.92 (d, 2H), 7.15–7.17 (d, 2H), 8.25–8.27 (d,
1H), 12.5 (b, 1H).
O-Methyl-l-homotyrosine (6a): In an Erlenmeyer
flask, compound 5a (2.00 g) was dissolved in potassium
phosphate buffer (80 ml, 100 mM), and the final pH was
adjusted to 8 using 6-M NaOH. Then, Aspergillus acylase
I (200 mg) was added to the mixture, and it was allowed
to incubate overnight at 37 °C, with a shaking speed of
250 rpm (C25, New Brunswick Scientific). The resulting
mixture was cooled, filtered, and washed with cold water
to give pure 6a as a crystalline solid. Yield: 573 mg (69 %).
1H NMR (D2O/NaOD) δ (ppm): 1.61–1.70 (m, 2H), 2.38–
2.42 (t, 2H), 3.03–3.06 (t, 1H), 3.62 (s, 3H), 6.76–6.78 (d,
2H), 7.05–7.07 (d, 2H).
L-Homotyrosine (1a): In a round-bottom flask, com-
pound 6a (0.1 g) was dissolved in concentrated HI. The
mixture was allowed to reflux overnight, and HI was
removed under reduced pressure. The residue was neu-
tralized using phosphate buffer (pH 7). The solvent was
removed under reduced pressure and a minimal amount
of cold water was added to the residue. The mixture was
O-Methyl-l-bishomotyrosine (6b) and L-bishomotyros-
ine (1b): Compounds 6b and 1b were obtained following
the same procedure described above for 6a and 1a. Bro-
mination of 2b using Appel’s salt afforded 3b as a clear
liquid in excellent yield (92 %). Subsequent alkylation
using DEAM provided 4b as a white solid (52 %). Ester
hydrolysis and decarboxylation of 4b was achieved in one
step by refluxing in aqueous NaOH to yield 5b as a white
solid in excellent yield (92 %). Enantioselective hydrolysis
of N-acetyl using Aspergillus acylase, as described above,
provided 6b in good yield (67 %). Deprotection using HI
gave compound 1b as a crystalline solid (69 % yield).
Enzymes and enzyme assays. Citrobacter freundii TPL
was obtained from Escherichia coli SVS370/pTZTPL
(Antson et al. 1993) and purified as previously described
(Chen et al. 1995a). The purified enzyme was stored as
aliquots at −78 °C and thawed immediately prior to use.
Enzyme assays were performed on a Cary 1 UV–vis-
ible spectrophotometer equipped with a thermoelectric
Peltier-controlled 6 × 6 cell changer. Enzyme activ-
ity was routinely determined using SOPC as previously
described (Chen et al. 1995b), by following the absorb-
ance decrease at λ = 370 nm (∆ε = 1.86 mM−1 cm−1).
SOPC was used as the substrate in inhibition assays for
TPL. All assays were conducted at 25 °C and in a total
volume of 600 µl. A typical assay contained potassium
phosphate buffer, pH 8.0 (50 mM), PLP (40 µM), and
varying concentration of SOPC and inhibitors. Experi-
mental velocities were analyzed, and Ki values were fit
to Eq. 4 by the FORTRAN program, COMPO, of Cle-
land (1979). Enzyme concentration was estimated from
the absorbance at λ = 278 nm (A1% = 8.37) (Kumagai
et al. 1970a) assuming a subunit mass of 51 kDa (Antson
et al. 1993).
v = V
max
∗ [S]/((Km(1 + [I]/Ki) + [S])
(4)
Rapid-scanning stopped-flow experiments Rapid-scan-
ning stopped-flow experiments were performed on an
RSM-1000 spectrophotometer (OLIS, Inc), equipped with
a stopped-flow cell of 1-cm pathlength, capable of collect-
ing up to 1000 scans per second with a dead time of ~2 ms.
Prior to performing kinetic experiments, the enzyme was
incubated with 0.5-mM PLP for 30 min at 30 °C. Subse-
quently, the enzyme was eluted through a short gel filtra-
tion column (PD-10, GE Healthcare) equilibrated with
phosphate buffer (pH 8.0, 50 mM) to remove excess PLP,
and rapid-scanning experiments were carried out in the
same buffer. The final concentration of the ligand was
5 mM. Rapid-scanning stopped-flow data were processed
1 3

Inhibition of tyrosine phenol-lyase by tyrosine homologues
Scheme 2 Synthesis of tyrosine homologues and O-methyl derivatives
Table 1 Ki values for tyrosine homologues
and analyzed by GlobalWorks software supplied by OLIS
(Matheson 1990).
Inhibitors
Ki (mM)
L-Homotyrosine (1a)
0.80 mM 0.20
1.50 mM 0.50
0.80 mM 0.17
1.04 mM 0.18
L-Bishomotyrosine (1b)
Results and discussion
O-Methoxy-L-homotyrosine (6a)
O-Methoxy-L-bishomotyrosine (6b)
Synthesis of tyrosine homologues and derivatives: The
synthesis and enzymatic resolution of 6b and 1b (Shimo-
higashi et al. 1976; Ueno et al. 1975), and the asymmetric
synthesis of 6a (Yamada et al. 1998) and 1a (Murashige
et al. 2011) have been previously reported. In our hands,
the most convenient method to obtain the optically active
Tyr homologues and their O-methyl derivatives was by fol-
lowing a modified method of Shimohigashi et al. (1976),
using diethyl acetamidomalonate, as described previously
(Scheme 2) (Do et al. 2014). 2-(4-Methoxyphenyl)etha-
nol (2a) and 3-(4-methoxyphenyl)propanol (2b) are com-
mercially available and were selected as starting materials.
As Appel’s salt is a milder reagent suitable for primary
alcohols, when bromination was carried out using this
reagent, we obtained excellent yield of 3. Alkylation of 3
using diethyl acetamidomalonate in sodium ethoxide pro-
ceeded to give 4 in modest yield. Subsequent hydrolysis
and decarboxylation of 4 was achieved in one step with
excellent yield of the N-acetyl derivatives, 5a and 5b, by
refluxing with two equivalents of NaOH in aqueous THF.
Enantioselective hydrolysis of 5 using Aspergillus acylase
(Chenault et al. 1989) was best carried out in phosphate
buffer at pH 8 instead of water, due to the low solubility
1 3

Q. Do et al.
Fig. 2 Spectra and time course
for reaction of TPL with 5 mM
L-homotyrosine (1a). a Spectra
at various times after mixing. b
Time courses at 500 nm (blue)
and 420 nm (black) (color figure
online)
Table 2 Rate constants
and absorption maxima of
intermediate formation of
homotyrosines with TPL
Compound
1/τ1
3.0 0.4
1/τ2
E
AL λmax (nm)
E
Q λmax (nm)
1a
1b
6a
6b
(6.2 0.5) × 10−1
387
390
394
387
505
496
499
496
5.0 0.3
5.3 0.3
2.39 0.21
(1.40 0.16) × 10−1
0.14 0.01
0.66 0.01
of compound 5 in water. Enzymatic resolution following
this method proceeded to give the optically active 6a and
6b in good yield. To the best of our knowledge, this is the
first report of the resolution of tyrosine homologues using
acylase. O-Dealkylation of 6a was previously reported to
proceed in concentrated HCl, by refluxing for three days
with optical activity remaining unaffected (Yamada et al.
1998). However, when this step was carried out in concen-
trated HI, we recovered similar yields for compound 1a and
1b within 15 h of reflux.
Enzyme inhibition: Our results indicated that com-
pounds 1a, 1b, 6a, and 6b are competitive inhibitors of
C. freundii TPL that exhibit Ki values in the range of
0.8–1.5 mM (Table 1), similar to that of l-phenylalanine
(2 mM). It is interesting to note that compounds 1a and
6a, homologues with extension by one methylene unit,
displayed slightly better inhibition than that of 1b and
6b, which were extended by two methylene units. Com-
petitive inhibitors of TPL, l-alanine and l-methionine,
form quinonoid complexes (Phillips et al. 2002; Milić
et al. 2008). While l-alanine forms quinonoid complexes
in both open and closed conformations, l-methionine
forms a quinonoid structure only in the closed conforma-
tion (Milić et al. 2008). It was previously suggested that
the phenolic OH of the substrate is hydrogen bonded to
Arg381 at the active site (Sundararaju et al. 1997), which
was later shown by the crystal structure of Y71F and
F448H mutant TPL with 3-F-l-tyrosine bound (Milić
et al. 2011). We were curious as to whether O-methyla-
tion would affect the inhibitory efficiency of the homo-
logues. Our results in Table 1 show no difference between
homologues 1a and 1b with their O-methyl derivatives
6a and 6b, suggesting that hydrogen bonding of the OH
is not essential for binding of the inhibitors evaluated in
this work. This also suggests that these homotyrosines do
not form closed conformations on binding to TPL, since
the hydrogen bonds to the OH are formed in the closed
conformation. Previously, we reported the evaluation of 2-
and 3-aza-l-tyrosines as inhibitors for TPL, with Ki values
of 135 µM and 3.4 mM, respectively (Watkins and Phil-
lips, 2001). Since 2-azatyrosine will exist as the phenolate
ion at neutral pH, we suggested that this is consistent with
binding of tyrosine to TPL as the phenolate. In compari-
son, the rationale behind our present design was to mimic
the elongated structure of the keto-quinonoid transition
state. However, the binding affinity of these compounds
for TPL is not as high as 2-aza-l-tyrosine, but better than
3-aza-l-tyrosine.
1 3

Inhibition of tyrosine phenol-lyase by tyrosine homologues
Scheme 3 PLP aldimine structures
Pre-steady-state kinetics: The absorbance spectra from
the rapid-scanning stopped-flow experiments share many
similarities between all compounds tested (Fig. 2 and
Supplemental Data). The reaction of tyrosine with TPL
was previously reported to give rise to two characteristic
absorption maxima at λmax = 418 and 502 nm, correspond-
ing to the formation of external aldimine and quinonoid
intermediates, respectively (Muro et al. 1978; Phillips et al.
2006). From our results, the presence of external aldimines
was observed in all cases, indicated by unusually blue-
shifted absorption maxima around λmax = 390 nm (Fig. 2;
Table 2). Also, the formation of quinonoid intermediates
was also observed for all compounds, evident by normal
quinonoid absorption maxima around λmax = 500 nm. Sur-
prisingly, the amplitude of the absorbance peak at 500 nm
for O-methyl-l-homotyrosine (6a) is much smaller than
L-homotyrosine (1a) and the bishomotyrosines (1b and 6b)
(Supplemental Data, Figure S1), even though there is lit-
tle difference in the values of Ki. These data demonstrate
that these inhibitors were bound to PLP at the active site
leading to formation of an equilibrating mixture of external
aldimine and quinonoid complexes. However, the low equi-
librium concentration of the quinonoid complexes implies
that they do not contribute significantly to the Ki values.
The absorption maxima of the aldimine intermediates for
these tyrosine homologues are blue-shifted slightly from
that reported (Muro et al. 1978; Phillips et al. 2006) in com-
parison with tyrosine as the substrate (Fig. 2). The strain that
exists in aldimines in the aminotransferase family is well
documented (Hayashi et al. 2003). Normally, the external
aldimine bonds are in plane with the PLP, with a hydrogen
bond formed between the iminium NH and the 3′-phenolate
of the PLP (Scheme 3), and they exhibit absorption max-
ima at about 425 nm. This was observed with TPL react-
ing with other substrate analogues, such as l-methionine
and l-alanine (Chen et al. 1995a,b; Phillips et al. 2002). The
aldimine can also be drawn as the ketoenamine resonance
structure (Scheme 3). However, if the imine is twisted out
of the plane of the pyridine ring, the intramolecular hydro-
gen bonding is weakened, as well as the resonance between
the iminium and the phenolate, resulting in the blue-shift in
the absorbance maximum. In some cases, an equilibrium
of the ketoenamine with an enolimine tautomer is present
(Scheme 3), but this is not seen in TPL. Our results sug-
gest that binding of the extended side chain of the tyrosine
homologues results in strained aldimines with the iminium
bond rotated out of plane of the PLP. This may be the result
of steric restraints imposed by the extended side chains of
these homologues. The energetic penalty resulting from the
strain may be a reason for the weaker than expected bind-
ing of these homologues. However, the relatively normal
absorption spectra and rates of formation of the quinonoid
complexes suggest that they are not strained, possibly due
to conformational changes of the enzyme upon quinon-
oid intermediate formation that relieve the strain. We note
that the structure of F448H mutant TPL complexed with
3-fluorotyrosine demonstrates formation of a closed active
site with a “tense” conformation of the quinonoid interme-
diate (Milić et al. 2011).
Conclusion
With promising results from our previous work on mech-
anism-based inhibitors of TIL, we extended our design
and rationale to TPL, since both enzymes share remark-
ably similar mechanistic details in the cleavage of their
physiological substrates. Also, it was also suggested that
the hydrogen bond from the phenol of the substrate with
Arg381 has an essential role in contributing to the Cα–Cβ–
Cγ bond angle strain that assisted in cleavage of the Cβ–Cγ
bond of the substrate. Based on the postulated mechanism
of TPL, we explored the effect of homologation of the Cα
position and deletion of the hydrogen bond donor site of
phenol from the physiological substrate, in the search for
mechanism-based inhibitors of TPL. Our results indicated
1 3

Q. Do et al.
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herein were in the range of 0.8–1.5 mM. Furthermore,
pre-steady-state kinetic evaluations confirmed binding of
these compounds at the active sites, forming an equilibrium
mixture of aldimines and quinonoid intermediates. These
results suggest that there may be conformational differ-
ences between TPL and TIL during catalysis.
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AA (2008) Insights into the catalytic mechanism of tyrosine
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D, Antson AA (2011) Crystallographic snapshots of tyrosine
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Acknowledgments Financial support to Q. D. during his PhD work
was provided by the University of Georgia.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Murashige R, Hayashi Y, Ohmori S, Torii A, Aizu Y, Muto Y, Murai
Y, Oda Y, Hashimoto M (2011) Comparisons of O-acylation and
Friedel-Crafts acylation of phenols and acyl chlorides and Fries
rearrangement of phenyl esters in trifluoromethanesulfonic acid:
effective synthesis of optically active homotyrosines. Tetrahe-
dron 67:641–649
Research involving human participants and/or animals This arti-
cle does not contain any studies with human participants or animals
performed by any of the authors.
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