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

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

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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. 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: 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) 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 à 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. References 1. 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