Yamskov1 1 Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia; 2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow,
Trang 1The mechanism of a-proton isotope exchange in amino acids catalysed
by tyrosine phenol-lyase
1 What is the role of quinonoid intermediates?
Nicolai G Faleev1, Tatyana V Demidkina2, Marina A Tsvetikova1, Robert S Phillips3and Igor A Yamskov1
1 Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia; 2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia;3Department 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 of 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 D2O, and we compared
the results with the rates of the isotopic exchange under the
same conditions We have found that, in theL-phenylalanine
reaction, the internal return of the a-proton is operative, and
allowing for its effect, the exchange rate is accounted for
satisfactorily Surprisingly, for the reaction withL -methio-nine, the enzymatic isotope 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 exchange; mechanism; a-proton; tyrosine phenol-lyase
Pyridoxal-P-phosphate (PLP)-dependent lyases displaying
broad substrate specificity 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 the natural (S)-configuration of amino acids
The characteristic 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-deuterated (S)-amino acids [5–7]
In the framework of the generally accepted notions of
mechanisms of PLP-dependent enzymes the mechanism of
the isotopic exchange 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 internal aldimine 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 external one (ES), which undergoes the abstraction of the a-proton by a certain enzyme group, leading to formation of a quinonoid intermediate (EA) The reversibility of the latter transfor-mation should lead in heavy water to the isotopic exchange
of the a-proton if the abstracted proton may be easily exchanged with the solvent However, the kinetics of 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 reaccondi-tions of PLP-dependent enzymes with amino acids and the rates of the enzymatic isotopic exchange for the same amino acids
To answer these questions, we studied in the present work the kinetics of quinonoid intermediate formation for the reactions of TPL withL-phenylalanine,L-methionine, and their a-deuterated analogs in D2O, and compared the results with the rates of the isotope exchange under the same conditions We have found that in the L-phenylalanine reaction the exchange of the 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 withL-methionine the enzymatic isotopic exchange proceeds much faster than it follows from the kinetic data for quinonoid intermediate formation This result allows us
to conclude 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: ngfal@ineos.ac.ru
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)
Trang 2reaction, while the exchange 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 freundiiin 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 concentration 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)Æmin)1under standard conditions
[12] Tryptophanase was prepared as described in [13]
a-Deuterated L-phenylalanine and L-methionine were
prepared by isotope exchange reactions in D2O using
tryptophanase as a catalyst: 0.45 gL-Phe was dissolved in
15 mL D2O, 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 D2O 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-deuteratedL-Phe The
procedure for preparation of a-deuterated L-Met was the
same, except initially 0.7 gL-Met was dissolved in 15 mL
D2O, and the time of incubation was 80 h
Stopped-flow measurements
Prior to performing rapid kinetic experiments, the stock
enzyme was incubated with 1 mM 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 0.1M potassium phosphate pH 8.7 For
experiments in D2O the enzyme solution was concentrated
to a minimal volume by ultrafiltration and diluted with
0.1Mpotassium phosphate in D2O 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 with an
RSM-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 0.1M potassium phosphate, pD (or pH) 8.7, were mixed with various concentrations of amino acids, and changes in absorbance at 500 nm were followed Rate constants were evaluated by exponential fitting using the
LMFTorSIFITprograms provided by OLIS The apparent rate constants from stopped-flow experiments were fitted to Eqn (1) usingENZFITTER(Elsevier) A representative exam-ple of a concentration dependence for quinonoid formation rates is given in Fig 1, and the calculated forward (kf) and
reverse (kr) 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) with K S , k f and
k given in Table 1.
Trang 3Isotope exchange experiments
The reaction with L-phenylalanine was run in 0.1M
potassium phosphate solution in D2O pD 8.7, containing
33.94 mM L-Phe, 0.1 mMpyridoxal-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 90° for 5 min to inactivate the enzyme The content of the
a-deuteratedL-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 mM and
2.64 unitsÆmL)1, respectively One-milliliter aliquots were
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 Mrof
51 000 [14]
Results and Discussion
Three-dimensional structures of TPLs 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 to Pletnev
et al.and Sundararaju et al [15,16], for TPL from
Citro-bacter freundii, Arg404 is the best candidate for the binding
of the a-carboxylate group of the substrate, 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 acid 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 cofactor plane [17], and shows that the pattern of binding is the same for a variety of amino acids It has been established [5] that for a number of amino acid inhibitors bearing nonbranched substituents without functional groups, the hydrophobicity of the side chain is the main factor controlling Ki 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 withL-phenylalanine andL-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 D2O 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.
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.
a
Maximum possible value.
Scheme 3.
Trang 4s¼ kf½S
The calculated kinetic parameters are presented in Table 1
The comparison of the rates of formal reprotonation (kr)
for the normal and a-deuterated substrates in D2O allowed
us to establish if there was any internal return of the
a-proton after its abstraction When internal return is really
operative the kr value for the nondeuterated substrate is
determined by a sum of two competing processes: the
protonation and the deuteration:
kr¼ akrðHÞþ ð1 aÞkrðDÞ 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 solvent molecules present 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 the reaction withL-phenylalanine the value of krfor
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 kr(D),
character-izing the deuteration process, corresponds to the krvalue for
the a-deuterated L-phenylalanine We assumed that the
value of kr(H), for the competing protonation process is
equal to the kr value for the reaction of nondeuterated
substrate in water The respective kinetic parameters,
determined in the present work are also presented in
Table 1, while the value of a, calculated by Eqn (2) is
presented in Table 2
For the reactions of bothL-methionine and a-deuterated
L-methionine in D2O the krvalues 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 reason to assume
the existence of any internal return, and the respective value
of a was assumed to be equal to zero (Table 2)
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 forms the aldimine bond with the carbonyl
group of the cofactor, PLP, in the holoenzyme structure
For the reaction of any nondeuterated substrate in D2O the
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 ofL-phenylalanine the observed value
of the internal return coefficient (a¼ 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 atom of the quinonoid intermediate should
go faster than the isotopic exchange of the proton in the
active site For the reaction of L-methionine, where 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 theL-phenylalanine reaction Thus, we may estimate the value of the isotope exchange rate from the protonated amino group as being considerably more than the kr(D) value for the reaction with L-methionine ( 0.01 s)1), and considerably less than that for the reaction withL-phenylalanine (3.1 s)1)
The overall process of isotopic exchange in amino acids may be described by the kinetic Scheme 2 The attainable degree of the exchange is determined by the isotopic purity
of D2O, which is high, and the 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 EAH, leading to aldimine ESD This implies that
as a result of this stage the a-proton, originally present in substrate SH, is irretrievably lost When this is taken into account, the quinonoid intermediates EAHand EADare formally nonidentical because for the former the protona-tion (internal return), leading to regeneraprotona-tion of the initial nondeuterated substrate is still possible, while the latter can
be only deuterated Thus, quinonoid intermediate EADis off the reaction pathway responsible for the principal transformation
Values of KSHand kf(H)correspond to KDand kffor the reaction of nondeuterated substrate, and KSD, kf(D) and
kr(D)are equal, respectively, to KD, kfand krfor the reaction
of deuterated substrate (Table 1) The values of Kmand kcat for the isotope exchange reaction may be described by Eqns (3) and (4)
Km¼ KSH½akrðHÞþ ð1 aÞkrðDÞ
kfðHÞþ akrðHÞþ ð1 aÞkrðDÞ
ð3Þ
Kcat¼ ð1 aÞkrðDÞkfðHÞ
kfðHÞþ akrðHÞþ ð1 aÞkrðDÞ ð4Þ The suggested mechanism implies also that the isotopic exchange reaction should be inhibited by the deuterated product The respective inhibition constant (Kp) is described
by Eqn (5)
Kp ¼ KSD
1þkfðDÞ
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 Km
Kp
¼ Kcat½E0t Km 1þ½S0
Kp
ln ½S0
½S0 ½P ð6Þ
In Figs 2 and 3 the theoretically expected dependencies for the reactions of TPL withL-phenylalanine andL -methio-nine, calculated with the use of the kinetic parameters presented in Table 2 are compared with the experimental data For the reaction of -phenylalanine, the experimental
Trang 5points at longer times lie somewhat below the theoretical
curve, which may be due to some inactivation of the 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 rate of isotopic exchange is mainly determined by
deuteration of the quinonoid intermediate
On the other hand, it is obvious from Fig 3 that for
reaction ofL-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 (kex¼ 0.37 s)1) is
by a factor of 22.5 faster than the highest possible kcatvalue 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 exchange rate Some comments are necessary as to the role played by the interaction of the side group ofL-methionine with Arg100 in the considered reactions ForL-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 different mechanism The real exchange rate (k*) corresponding to this mechanism should be much more than the observed one, because in the experimental condi-tions most of the enzyme is bound in the inactive quinonoid intermediate For a simple kinetic scheme (Scheme 4) the observed exchange rate may be described
by Eqn (7):
kex¼ k
1þkf
kr
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] have shown for the reaction of cytosolic aspartate aminotransferase that the proton transfer from the Ca to the C4, position of the cofactor occurs as a concerted 1,3-prototropic shift, whereas the quinonoid intermediate, although it is formed, is a dead-end complex Phillips et al [22] provided evidence that elimination of indole in the tryptophanase reaction is realized by a concerted SE2 mechanism, involving simul-taneous protonation of the C3atom of the indole moiety and breakdown of the C3-Cbbond Tai and Cook [23] have shown that a concerted anti-E2mechanism is realized for the elimination of acetate from O-acetyl-L-serine, catalyzed
Scheme 4.
Fig 3 Isotopic exchange of L -methionine under the action of TPL The
reaction was run in 0.1 M potassium phosphate buffer in D 2 O pD ¼
8.7, containing 95.23 m M L -Met, 0.1 m M PLP, and 2.64 unitsÆmL)1
TPL j, Experimental data; solid line, the experimental curve
calcu-lated using Eqn (6) and kinetic parameters from Table 2.
Fig 2 Isotopic exchange of L -phenylalanine under the action of TPL.
The reaction was run in 0.1 M potassium phosphate buffer in D 2 O
pD ¼ 8.7, containing 33.94 m M L -Phe, 0.1 m M PLP, and 1.27 unitsÆ
mL)1TPL d, Experimental data; solid line, the experimental curve
calculated using Eqn (6) and kinetic parameters from Table 2.
Trang 6by O-acetylserine sulfhydrylase By analogy with these
findings, a concerted mechanism of isotopic exchange may
be considered as a possible alternative The concerted
mechanism, involving the Lys257 amino group and the
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 (D2O) molecule may ensure the
formation of a favorable six-membered transition state
(Fig 4) Such a mechanism might 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 tuning between the external aldimine and
the active site structures, probably resulting in some
deviation from the geometry optimal for the abstraction
of the a-proton For the reaction of TPL withL
-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 than for the reaction with 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-proton This
distortion of the proper spatial organization of the active
site is, probably, more favorable for the formation of
the six-membered transition state From comparison of
kf¼ 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 40 than the normal a-proton
abstraction in the complex of TPL withL-methionine
Acknowledgments
This research was supported by grants from the Russian Foundation
for Basic Researches (04-04-49370) to N.G.F and Fogarty
Interna-tional Center (TW00106) to R.S.P and T.V.D.
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