Tài liệu Báo cáo khoa học: The mechanism of a-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase doc

The mechanism of a-proton isotope exchange in amino acids catalysed
by tyrosine phenol-lyase
1
What is the role of quinonoid intermediates?
Nicolai G. Faleev
1
, Tatyana V. Demidkina
2
, Marina A. Tsvetikova
1
, Robert S. Phillips
3
and Igor A. Yamskov
1
1
Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia;
2
Engelhardt Institute of
Molecular Biology, Russian Academy of Sciences, Moscow, Russia;
3
Department of Chemistry, Department of Biochemistry and
Molecular Biology, and Center for Metalloenzyme Studies, University of Georgia, Athens, GA, USA
To shed light on the mechanism o f isotopic exchange of
a-protons in amino acids catalyzed by pyridoxal phosphate
(PLP)-dependent enzymes, we studied the kinetics of
quinonoid intermediate formation for the reactions of
tyrosine phenol-lyase with
L
-phenylalanine,
L
-methionine,

and their a-deuterated analogues in D
2
O, and we compared
the r esults with the rates of the isotopic exchange under the
same conditions. We have found that, in the
L
-phenylalanine
reaction, the internal return o f the a-proton is operative, and
allowing for its effect, the exchange rate is accounted for
satisfactorily. Surprisingly, for the reaction with
L
-methio-
nine, the enzymatic i sotope exchange went much faster than
might be predicted from the kinetic data for quinonoid
intermediate formation. This result allows us to suggest the
existence of an alternative, possibly concerted, mechanism of
a-proton exchange.
Keywords: amino acids; isotopic e xchange; mechanism;
a-proton; tyrosine phenol-lyase.
Pyridoxal-P-phosphate (PLP)-dependent lyases displaying
broad substrate s pecificity are able to catalyze stereospecific
isotope exchange of a-protons of various amino acids [1–4]
including both real substrates and reversible competitive
inhibitors, which do not change their chemical identities
under the action of the enzyme. The exchange is usually
performed in heavy water, and proceeds with a complete
retention of t he natural (S)-configuration of amino acids.
The c haracteristic PLP-dependent enzymes in this respect
are tyrosine phenol-lyase (TPL) (EC 4.1.99.2), tryptophan
indole-lyase (EC 4.1.99.1), and

L
-methionine-c-lyase
(EC 4.4.1.11). These enzymes are used as very effective
biocatalysts for preparation of enantiomerically pure
a-de uterated (S)-amino acids [5–7].
In the framework of the generally accepted notions of
mechanisms of PLP-dependent enzymes the mechanism of
the i sotopic e xchange traditionally is considered to be
associated with formation of quinonoid intermediates
(Scheme 1). In the holoenzymes (E) the cofactor PLP is
bound in the active site as an Ôinternal aldimineÕ with an
e-amino group of a definite lysine residue. As a result of
interaction with an amino acid substrate, or inhibitor, the
internal aldimine (E) is substituted by an ÔexternalÕ one (ES),
which undergoes the abstraction of the a-proton by a
certain enzyme group, leading to formatio n of a Ôquinonoid
intermediateÕ (EA). The reversibility of the latter transfor-
mation should lead in heavy water to the isotopic exchange
of the a-proton if t he abstracted proton may be easily
exchanged with t he solvent. However, the kinetics o f
quinonoid formation was examined until now only in water
solutions [8–11], while measurements in heavy water, in
conditions identical to those of the isotopic exchange, were
not performed. No attempts to quantitatively estimate the
rates of the exchange of the abstracted proton in the active
site have been reported. We have noted earlier [8] that no
direct correlation was observed between the amount of the
quinonoid intermediate formed under steady-state condi-
tions in reactions of PLP-dependent enzymes with amino
acids and the rates of the enzymatic isotopic exchange for

thesameaminoacids.
To answer these questions, we studied in the present work
the kinetics of quinonoid intermediate formation for the
reactions of TPL with
L
-phenylalanine,
L
-methionine, and
their a-deuterated analogs in D
2
O, and compared the results
with the rates of the i sotope exchange under t he same
conditions. We h ave found that in the
L
-phenylalanine
reaction the exchange o f t he abstracted proton in the active
site proceeds more slowly than the reprotonation reaction,
leading to a considerable internal return of the a-proton.
Allowing for this effect, the rate of the enzymatic isotopic
exchange is accounted for satisfactorily. Surprisingly, for the
reaction with
L
-methionine the enzymatic isotopic exchange
proceeds much faster than it follows from the kinetic data
for quinonoid intermediate formation. This result allows us
to co nclude that the quinonoid is a dead-end complex in this
Correspondence to N. G. Faleev, Nesmeyanov Institute of Organo-
element Compounds, Russian Academy of Sciences, 28 Vavilov Street,
Moscow, 119991, Russia. Fax: +95 1355085, Tel.: +95 1356458,
E-mail:

Abbreviations: PLP, pyridoxal-P-phosphate; TPL, tyrosine phenol-
lyase; SOPC, S-o-nitrophenyl-
L
-cysteine.
Enzymes: tyrosine phenol-lyase (EC 4.1.99.2); tryptophan indole-lyase
(EC 4.1.99.1);
L
-methionine-c-lyase (EC 4.4.1.11); aspartate amino-
transferase (EC 2.6.1.1).
(Received 6 July 2004, revised 7 September 2004,
accepted 8 October 2004)
Eur. J. Biochem. 271, 4565–4571 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04428.x
reaction, while the e xchange of the a-proton is realized by
an alternative, possibly concerted, mechanism.
Materials and methods
Materials
TPL was obtained from Escherichia coli SVS370 cells con-
taining plasmid pTZTPL, which contains the tpl gene from
Citrobacter freundii in pTZ18U (US Biochemical, Cleveland,
OH, USA)
2
, as described [10]. The enzyme obtained was
apparently homogeneous and had specific activity of
4.91 unitsÆmg
)1
. The co ncentration of the active enzyme was
determined by activity measurements, assuming that the pure
enzyme enzyme had a maximum specific activity of 6 unitsÆ
mg
)1

[10]. One unit of activity was determined as amount of
enzyme catalyzing the decomposition of 1 lmol S-o-nitro-
phenyl-L-cysteine (SOPC)Æmi n
)1
under standard conditions
[12]. Tryptophanase was prepared as described in [13].
a-Deu terated
L
-phenylalanine and
L
-methionine were
prepared by isotope exchange reactions in D
2
Ousing
tryptophanase as a catalyst: 0.45 g
L
-Phe was dissolved in
15 mL D
2
O, 3 mg of tryptophanase was added, the pH of
the solution measured with glass electrode was adjusted to
8.6 by adding KOH solution in D
2
O. After incubation for
68 h the mixture was analyzed by PMR. The degree of
a-proton exchange was shown to be > 98%. The solution
was heated to 95 °C to inactivate the enzyme, and then
evaporated to dryness, and the residue was recrystallized
from water/ethanol to obtain pure a-deuterated
L

-Phe. The
procedure for preparation of a-deuterated
L
-Met was the
same, except initially 0.7 g
L
-Met was dissolved in 15 mL
D
2
O, and the time of incubation was 80 h.
Stopped-flow measurements
Prior to performing rapid kinetic experiments, the stock
enzyme was incubated with 1 m
M
pyridoxal-P for 1 h at
30 °C at pH 7.0 and then separated from excess pyridoxal-P
using a short desalting column (PD-10, Pharmacia) equili-
brated with 0.1
M
potassium phosphate pH 8.7. For
experiments in D
2
O the enzyme solution was concentrated
to a minimal volume by ultrafiltration and diluted with
0.1
M
potassium phosphate in D
2
O pD 8.7. To determine
pD values an allowance was made for the isotope effect of

the glass electrode (0.4). The concentration and dilution
procedure was repeated three times. Rapid-scanning
stopped-flow kinetic data were obtained w ith an R SM-
1000 instrument from OLIS, Inc. This instrument has a
dead time of % 2 ms, and is capable of collecting spectra in
the visible region from 300 to 600 nm at 1 kHz. The enzyme
solutions in 0.1
M
potassium phosphate, pD (or pH) 8.7,
were mixed w ith various concentrations of amino acids, and
changes in absorbance a t 500 nm were followed. Rate
constants were evaluated by exponential fitting using the
LMFT
or
SIFIT
programs provided by OLIS. The apparent
rate constants from stopped-flow experiments were fi tted to
Eqn (1) using
ENZFITTER
(Elsevier). A representative exam-
ple of a concentration dependence for quinonoid formation
rates is given in Fig. 1, and t he calculated ÔforwardÕ (k
f
)and
Ôre verseÕ (k
r
) rate constants are presented in Table 1.
Assuming that isotope exchange reactions were described
by Scheme 2
3

the respective kinetic parameters were calcu-
lated using Eqns (3–5), and are presented in Table 2.
Scheme 1.
Fig. 1. The concentration dependence for quinonoid formation rates for
the reaction of TPL with a-deuterated
L
-phenylalanine in D
2
O. d,
Experimental data; solid line, calculated fit to Eqn (1) w ith K
S
, k
f
and
k
r
given in Table 1.
4566 N. G. Faleev et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Isotope exchange experiments
The reaction with
L
-phenylalanine was run in 0.1
M
potassium phosphate solution in D
2
O pD 8.7, containing
33.94 m
ML
-Phe, 0.1 m
M

pyridoxal-P, and 1.27 unitsÆmL
)1
TPL. Aliquots (1 mL) of the reaction mixture were
withdrawn after 71, 125, 265, 381 and 490 min and heated
at 9 0° for 5 min to inactivate the enzyme. The content o f the
a-de uterated
L
-Phe was determined by PMR. The reaction
with
L
-Met was run under the same conditions the
concentration of
L
-Met and TPL being 95.23 m
M
and
2.64 unitsÆmL
)1
, respectively. One-millili ter aliquots w ere
withdrawn after 108, 250, 360, 568, and 754 min and treated
as above. The theoretical values of isotopic exchange rates
were calculated, based on the assumption that the number
of operative active sites participating in reactions of SOPC
decomposition and isotope exchange was the same (one
active siteÆper subunit), given that a subunit had an M
r
of
51 000 [14].
Results and Discussion
Three-dimensional structures of T PLs from different micro-

bial sources have been established by X-ray studies [14–16].
It was shown that the cofactor, PLP, occupies a strictly
determined position in the active site. According t o Pletnev
et al. and Sundararaju et al. [15,16], for TPL from Citro-
bacter freundii, Arg404 is the best candidate for t he binding
of the a-carboxylate group of the s ubstrate, when the
external aldimine is formed. The anchoring of a-carboxylate
and a-amino group in the external aldimine defines
automatically the positions of the a-proton and the side
chain of any bound amino ac id. The lability of the a-proton
observed for a large number of amino acids [5] under the
action of TPL gives evidence for the orthogonal orientation
of the a-proton with respect to the cofa ctor plane [17], and
shows that t he pattern of binding is the same for a variety of
amino acids. It has been established [5] that for a number of
amino acid i nhibitors bearing nonbranched substituents
without functional groups, the hydrophobicity of the side
chain is the main factor controlling K
i
. Amino acids that
contain nucleophilic side chains (
L
-aspartic acid,
L
-homo-
serine,
L
-methionine,
L
-glutamic acid) exhibit enhanced

affinities for the enzyme. It was supposed that these
nucleophilic substituents interact with an electrophilic group
in the active site [5]. Evidence was presented by Mouratou
et al. [18] that Arg100 occupies a suitable position to per-
form such an interaction. In the present work we examined
the mechanisms of isotopic exchange of a-proton catalyzed
by TPL in reactions with
L
-phenylalanine and
L
-methionine
which may be considered as typical representatives of the
two groups of amino acid inhibitors mentioned above.
The interaction of
L
-phenylalanine,
L
-methionine, and
their a-deuterated analogs with TPL in D
2
O was charac-
terized by the appearance of quinonoid intermediates,
absorbing at % 500 nm. The kinetic curves were satisfac-
torily fitted by single exponentials, as was observed previ-
ously for the respective reactions in water [9]. The
concentration dependencies of the observed rates are well
described by Eqn (1); consequently, the reactions obey the
general Scheme 3 [19], where complex ES is the external
aldimine, and complex EA is the quinonoid intermediate.
Table 1. Kinetic parameters of reversible quinonoid formation for the reactions of TPL with

L
-phenylalanine,
L
-methionine, and their deuterated
analogs.
Substrate Solvent K
S
(m
M
) k
f
(s
)1
) k
r
(s
)1
)
L
-Phenylalanine
2
H
2
O 1.14 ± 0.11 14.9 ± 0.35 3.1 ± 0.24
[a-
2
H]-
L
-Phenylalanine
2

H
2
O 0.81 ± 0.39 1.52 ± 0.17 1.28 ± 0.15
L
-Phenylalanine H
2
O 0.92 ± 0.11 14.1 ± 0.4 7.46 ± 0.45
L
-Methionine
2
H
2
O 35.8 ± 1.1 5.85 ± 0.08 0.0083 ± 0.007
[a-
2
H]-
L
-Methionine
2
H
2
O 19.5 ± 1.6 0.62 ± 0.01 0.0065 ± 0.0046
Scheme 2.
Table 2. The calculated kinetic parameters for the isotopic exchange
reactions of
L
-phenylalanine and
L
-methionine catalyzed by TPL in
comparison with the kinetic parameters of TPL reaction with its natural

substrate.
Substrate a K
m
(m
M
) k
cat
(s
)1
) K
p
(m
M
)
L
-Phenylalanine 0.294 0.196 0.748 0.370
L
-Methionine 0 0.0915 0.015
a
0.340
L
-Tyrosine – 0.2 [10] 3.5 [10] –
a
Maximum possible value.
Scheme 3.
Ó FEBS 2004 Enzymatic a-proton exchange in amino acids (Eur. J. Biochem. 271) 4567
k
obs
¼
1

s
¼
k
f
½S
K
S
þ½S
þ k
r
ð1Þ
The calculated kinetic parameters are presented in Table 1.
The comparison of the rates of f ormal ÔreprotonationÕ (k
r
)
for the normal and a-deuterated substrates in D
2
O allowed
us to establish if t here was any internal return of the
a-prot on after its abstraction. When internal return is really
operative the k
r
value for the nondeuterated substrate is
determined by a sum of two c ompeting processes: the
protonation and the deuteration:
k
r
¼ ak
rðHÞ
þð1 À aÞk

rðDÞ
ð2Þ
The relative contributions of these processes are described
by a partition coefficient a, which is determined by: (a) the
rates of the isotopic exchange between the enzyme func-
tional group having abstracted the a-proton, and existing as
a conjugate acid, and surrounding groups, capable of
isotopic exchange, and s olvent molecules p resent in the
active site; (b) the statistical factor taking account of the
ratio of protons and deuterons on the considered group
when the latter is polyprotic; (c) the degree of restriction of
the free rotation of the considered group in the active site.
For t he reaction with
L
-phenylalanine the value of k
r
for
nondeuterated substrate is more than for the a-deuterated
one by a factor of 2.4. This indicates the presence of a
considerable internal return. The value of k
r(D)
, character-
izing t he deuteration process, corresponds to the k
r
value f or
the a-deuterated
L
-phenylalanine. We a ssumed that t he
value of k
r(H)

, for the competing protonation process is
equal to the k
r
value for the reaction of nondeuterated
substrate in w ater. The respective kinetic parameters,
determined in the p resent work are also presented in
Table 1, w hile the value of a, calculated b y Eqn (2) is
presented in Table 2.
For the reactions of both
L
-methionine and a-deuterated
L
-methionine in D
2
Othek
r
values are very small, and could
be determined only with high experimental errors. In the
limits of these errors, the rates for the normal and deuterated
substrates did not differ, thus, there was no r eason to assume
the existence of any internal return, and the respective value
of a wasassumedtobeequaltozero(Table2).
According to X-ray data [15,16] the abstraction of the
a-proton is most probably effected by the e-amino group of
lysine 257, which f orms the aldimine bond with the carbonyl
group of the cofactor, PLP, in the holoenzyme structure.
For the reaction of any nondeuterated substrate in D
2
Othe
amino group, after the proton abstraction, should exist as a

conjugate acid, bearing a positive charge and containing
two deuteriums and one hydrogen at the nitrogen atom. If
rotation around the C–Nbond is not restricted, the statis-
tical factor for the internal return of the proton is equal to
0.33. For the reaction of
L
-phenylalanine the observed value
of the internal return coefficient (a ¼ 0294) is only slightly
less. Consequently, it is reasonable to conclude that the
transfer of the proton (or deuteron) from the amino group
to the a-carbon ato m of the quinonoid intermediate should
go faster than the isotopic exchange of the proton in the
active site. For the reaction of
L
-methionine, w here no
internal return is observed, on the contrary, the isotopic
exchange goes faster, which seems natural because the
deuteration of the quinonoid intermediate proceeds much
slower than in the
L
-phenylalanine reaction. Thus, we may
estimate the va lue of the isotope exchange rate from the
protonated amino group as being considerably more than
the k
r(D)
value for the reaction with
L
-methionine
(% 0.01 s
)1

), and considerably less than that for the reaction
with
L
-phenylalanine (3.1 s
)1
).
The overall process of isotopic exchan ge in amino acids
may be described by the kinetic Scheme 2. The attainable
degree of the exchange is determined by the isotopic purity
of D
2
O, which is high, and t he equilibrium isotope effect,
which favors the exchange because the fractionation factor
is greater than one for an O–D/C–D equilibrium. Taking
these considerations into account the whole reaction may be
assumed to be irreversible. In the frames of the suggested
scheme the principal irreversible stage is the deuteration of
quinonoid EA
H
, leading to aldimine ES
D
. This implies that
as a result of this stage the a-proton, originally present in
substrate S
H
, is irretrievably lost. When this is taken into
account, the quinonoid intermediates EA
H
and EA
D

are
formally nonidentical because for the former the protona-
tion (internal return), leading to regeneration of the initial
nondeuterated substrate is still possible, while the latter can
be only deuterated. Thus, quinonoid intermediate EA
D
is
off the reaction pathway responsible for the principal
transformation.
Values of K
SH
and k
f(H)
correspond to K
D
and k
f
for the
reaction of nondeuterated substrate, and K
SD
, k
f(D)
and
k
r(D)
are equal, respectively, to K
D
, k
f
and k

r
for the reaction
of deuterated substrate (Table 1). The values o f K
m
and k
cat
for the isotope exchange reaction may be described by Eqn s
(3) and (4).
K
m
¼
K
SH
½ak
rðHÞ
þð1 À aÞk
rðDÞ

k
fðHÞ
þ ak
rðHÞ
þð1 À aÞk
rðDÞ
ð3Þ
K
cat
¼
ð1 À aÞk
rðDÞ

k
fðHÞ
k
fðHÞ
þ ak
rðHÞ
þð1 À aÞk
rðDÞ
ð4Þ
The suggested mechanism implies also t hat the isotopic
exchange reaction should be inhibited by the deuterated
product. The respective i nhibition constant (K
p
) is described
by Eqn (5).
K
p
¼
KS
D
1 þ
k
fðDÞ
k
rðDÞ
ð5Þ
The theoretical kinetic parameters calculated in this way are
presented in Table 2.
For enzymatic reactions where inhibition by product is
observed the dependence of product concentration on time

may be described by the Foster–Niemann equation [20]:
½P 1 À
K
m
K
p

¼ K
cat
½E
0
t À K
m
1 þ
½S
0
K
p

ln
½S
0
½S
0
À½P
ð6Þ
In Figs 2 and 3 the theoretically expected dependencies for
the reactions of TPL with
L
-phenylalanine and

L
-methio-
nine, calculated with t he use o f t he kinetic p arameters
presented in Table 2 are compared with the experimental
data. For the reaction of
L
-phenylalanine, the experimental
4568 N. G. Faleev et al. (Eur. J. Biochem. 271) Ó FEBS 2004
points at longer times lie somewhat below the theoretical
curve, which may be due to some inac tivation o f t he enzyme
during the reaction. In general, however, the deviations of
the experimental values from the calculated ones are not
significant. We believe therefore that for this reaction the
traditional mechanism of isotopic exchange, involving the
formation of a quinonoid species as a principal intermediate
structure, agrees satisfactorily with the experimental results.
The r ate o f isotopic e xchange i s mainly determined b y
deuteration of the quinonoid intermediate.
On the other hand, it is obvious from Fig. 3 that for
reaction of
L
-methionine the experimental data can in no
way be reconciled with the theoretically expected results.
The experimental values are much higher than the calcula-
ted ones, and the initial rate of exchange (k
ex
¼ 0.37 s
)1
)is
by a f actor of 2 2.5 faster than the highest possible k

cat
value.
Thus, the quinonoid intermediate, which is formed in the
L
-methionine reaction as a predominant structure, cannot
be considered as a principal intermediate in the isotopic
exchange process, because the rate of its deuteration is too
low as compared to the observed isotopic e xchange rate.
Some comments are necessary as to the role played by the
interaction of the side group of
L
-methionine with Arg100 in
the considered reactions. For
L
-aspartic acid the interaction
of the distal carboxylic group with Arg100 takes place in
the quinonoid intermediate structure [18], but not in the
external aldimine. The observed predominant formation of
the quinonoid intermediate in the reaction of TPL with
L
-methionine gives evidence for the presence of a similar
interaction of sulfur atom with Arg100, and the observed
very low rate of reprotonation evidently reflects the
stabilization of the quinonoid intermediate by this inter-
action. We have to conclude, therefore, that the isotopic
exchange of a-proton should for the most part be effected
by a d ifferent mechanism. The real exchange r ate ( k*)
corresponding to this mechanism should be much more
than the observed one, because in the experimental condi-
tions most of the enzyme i s bound in the ÔinactiveÕ

quinonoid intermediate. For a simple kinetic scheme
(Scheme 4) the observed exchange rate may be described
by Eqn (7):
k
ex
¼
k
Ã
1þ
k
f
k
r
ð7Þ
and the k* value estimated in this way should be equal to
230–240 s
)1
.
Considering alternative mechanisms of the isotopic
exchange we should note that although numerous exam-
ples of apparent stepwise mechanisms in reactions of PLP-
dependent enzymes are known, in some cases an interesting
tendency to utilize concerted mechanisms was observed.
Julin and Kirsch [21] h ave shown for the reaction o f
cytosolic aspartate aminotransferase that the proton
transfer from the C
a
to the C
4
, position of the cofactor

occurs as a concerted 1 ,3-prototropic shift, w hereas the
quinonoid intermediate, although it is formed, is a dead-
end complex. P hillips et al. [22] p rovided e vidence t hat
elimination of indole in the tryptophanase reaction is
realized by a concerted S
E
2 mechanism, involving simul-
taneous protonation of the C
3
atom of the indole moiety
and breakdown of the C
3
-C
b
bond. Tai and Cook [23] have
shown that a concerted anti-E
2
mechanism is realized for
the elimination of acetate from O-acetyl-
L
-serine, catalyzed
Scheme 4.
Fig. 3. Isotopic e xchange of
L
-methionine under the action of TPL. The
reaction was run in 0.1
M
potassium phosphate buffer in D
2
OpD¼

8.7, containing 95.23 m
ML
-Met, 0.1 m
M
PLP, and 2.64 unitsÆmL
)1
TPL. j, Experimental data; solid line, t he experimental curve calcu-
latedusingEqn(6)andkineticparametersfromTable2.
Fig. 2. Isotopic exchange of
L
-phenylalanine under the action of TPL.
Thereactionwasrunin0.1
M
potassium phosphate buffer in D
2
O
pD ¼ 8.7, containing 33.94 m
ML
-Phe, 0.1 m
M
PLP, and 1.27 unitsÆ
mL
)1
TPL. d, Experimental data; solid line, the experimental curve
calculated using Eqn (6) and kinetic parameters from Table 2.
Ó FEBS 2004 Enzymatic a-proton exchange in amino acids (Eur. J. Biochem. 271) 4569
by O-acetylserine sulfhydrylase. By analogy w ith these
findings, a concerted mechanism of isotopic exchange may
be considered as a possible alternative. The concerted
mechanism, involving t he Lys257 amino group and t he

C-H bond of the external aldimine implies formation of a
four-membered cyclic transition state, which energetically
is not favorable. We may reasonably suggest, however, that
involvement of a water (D
2
O) molecule may ensure the
formation of a favorable six-membered transition state
(Fig. 4). Such a mechanism m ight be facilitated by a
preliminary formation of a hydrogen bond between the
Lys257 amino group and a water molecule providing a
favorable mutual orientation of the amino group, the
water, and the a-proton of the external aldimine. The
formation of a symmetrical six-membered transition state
implies a subtle ÔtuningÕ between the external aldimine and
the active site structures, probably r esulting in some
deviation from the geometry optimal for the abstraction
of the a-proton. For the reaction of TPL with
L
-methio-
nine the rate of abstraction of the a-proton, leading to
formation of the quinonoid intermediate, is less by a factor
of 2.5 t han f or the r eaction w ith
L
-phenylalanine. The
observed retardation shows that orientation of the amino
group of Lys257 with respect to the a- proton is, in fact, not
quite favorable for the abstraction of a-p roton. T his
distortion of the ÔproperÕ spatial organization of the active
site is, p robably, more favorable f or the formation of
the s ix-membered transition s tate. F rom comparison of

k
f
¼ 5.85 s
)1
(Table 1) and k* ¼ 230–240 s
)1
it follows
that the putative concerted isotopic exchange should go
faster by a factor of 4 0 t han the ÔnormalÕ a-proton
abstractioninthecomplexofTPLwith
L
-methionine.
Acknowledgments
This research was supported by grants from the Russian Foundation
for Basic Researches (0 4-04-49370 ) to N.G.F. and Fogarty Interna-
tional Center (TW00106) to R.S.P. and T.V.D.
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