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Tiêu đề The mechanism of a-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase
Tác giả Nicolai G. Faleev, Tatyana V. Demidkina, Marina A. Tsvetikova, Robert S. Phillips, Igor A. Yamskov
Trường học University of Georgia
Chuyên ngành Biochemistry and molecular biology
Thể loại Báo cáo khoa học
Năm xuất bản 2004
Thành phố Moscow
Định dạng
Số trang 7
Dung lượng 170,58 KB

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Yamskov1 1 Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia; 2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow,

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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 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)

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reaction, 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.

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Isotope 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.

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s¼ 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 5

points 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.

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by 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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the formation of a six-membered transition state.

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