1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 117984, Russia 2 Departments of Chemistry and of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556, USA 3 Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA and 4 Nesmeyanov Institute of Organo-Element Compounds, Russian Academy of Sciences, 28 Vavilov Street, Moscow 117813, Russia
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Abstract |
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Keywords: ß-elimination/lyases/mutagenesis/pyridoxal 5' -phosphate/tyrosine
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Introduction |
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In addition to L-tyrosine, L-serine, S-methyl-L-cysteine (Kumagai et al., 1970), S-ethyl-L-cysteine, S-(o-nitrophenyl)-L-cysteine, O-benzoyl-L-serine (Phillips, 1987
) and ß-chloro-L-alanine act as substrates for ß-elimination, with formation of ammonium pyruvate. TPL also catalyzes the reverse reaction of ß-elimination (Enei et al, 1972
; Yamada et al., 1972
) and the ß-replacement reactions between substrates for ß-elimination and phenol derivatives (Equation 2)
(Kumagai et al., 1969
; Ueno et al., 1970
) as well as transamination side reactions (Demidkina et al., 1987
).
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The generally accepted mechanism of TPL catalysis is presented in Scheme 1.
Pyridoxal-P-dependent enzymes catalyze numerous reactions involved in amino acid metabolism. These reactions, although chemically different, are all performed with the use of the same cofactor, thus pyridoxal-P-dependent enzymes are very attractive objects to study the role of the protein matrix in providing substrate and reaction specificity. To date, three-dimensional structures have recently been determined for several pyridoxal-P-dependent enzymes belonging to different classes (Jansonius, 1998). For TPL three-dimensional structures have been recently determined for apo- and holoenzyme from Citrobacter freundii (Antson et al., 1993
) and Erwinia herbicola (Pletnev et al., 1997
) and for two enzymeinhibitor complexes (Pletnev et al., 1997
; Sundararaju et al., 1997
). Crystallographic data and site-directed mutagenesis studies have allowed us to clarify the roles of two catalytic residues (Arg381 and Tyr71) (Chen et al., 1995a
; Sundararaju et al., 1997
). In the present work, we studied the role of the asparagine 185 residue. Although this residue does not directly participate in catalysis, it is invariant in all known primary structures of TPL and of closely related tryptophan indole-lyase from several organisms (Chen et al., 1995a
) and it may be aligned with the cofactor-binding asparagine 194 in aspartate aminotransferase, the most thoroughly studied pyridoxal-P-dependent enzyme (Yano et al., 1993
). To shed light on the role of Asn185 in TPL catalysis we prepared a mutant TPL in which Asn185 is replaced with alanine and compared its properties with those of the wild-type TPL. The results obtained demonstrate that Asn185 plays a role in providing effective substrate binding and in catalysis through stabilization of a quinonoid intermediate.
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Materials and methods |
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Lactate dehydrogenase from rabbit muscle, pyridoxal-P and NADH were purchased from United States Biochemical (USB), as were L-tyrosine, S-benzyl-L-cysteine and S-ethyl-L-cysteine. ß-Chloro-L-alanine, L-phenylalanine, L-methionine, L-aspartic acid and L-homoserine were obtained from Sigma. S-Methyl-L-cysteine was a product of ICN Biochemicals.S-(o-Nitrophenyl)-L-cysteine (SOPC) was prepared as described previously (Phillips et al., 1989). 3-Fluoro-L-tyrosine was prepared by enzymatic synthesis (Phillips et al., 1990
). [
-2H]-3-Fluoro-L-tyrosine was made by performing the enzymatic synthesis in 2H2O as described previously for L-tyrosine (Kilk and Phillips, 1988). [
,ß,ß-2H3]-3-Fluoro-L-tyrosine was prepared as described by Faleev et al. (1996).
Preparation of N185A TPL
Oligonucleotide-directed site-specific mutagenesis was carried out with the use of plasmid pTZTPL, which contains the tpl gene from C.freundii in pTZ18U (US Biochemical) (Antson et al., 1993). Single-stranded DNA was prepared with the aid of M13K07 helper phage (Vieira and Messing, 1987
). Site-directed mutagenesis was performed by the method of Taylor et al. (1985) using the Sculptor in vitro mutagenesis kit from Amersham. To perform the required Asn185Ala replacement the mutagenetic oligonucleotide 5'-AGTCACGGTTGCCCTCGCAGGCGG-3 was prepared. The resulting plasmids were screened by dideoxy sequencing (Sanger et al., 1977
) in the mutation region using Sequenase (US Biochemical.) and a primer complementary to nucleotides 10501073 in the tpl gene sequence defined by Antson et al. (1993). The plasmid carrying N185A change was designated pTZTPL N185A. Escherichia coli SVS370 cells were used as the host for the plasmid. The cells were grown and the enzyme was purified as described previously (Chen et al., 1995b
). Wild-type TPL was purified from E.coli SVS370 cells containing plasmid pTZTPL by the same procedure.
Enzyme assays
Enzyme activity was routinely measured with 0.6 mM S-(o-nitrophenyl)-L-cysteine (SOPC) in 50 mM potassium phosphate, pH 8.0, at 25°C (Phillips, 1987) by following the decrease in absorbance at 370 nm (
= 1.86x103 l/mol.cm). One unit of activity was determined as the amount of enzyme catalyzing the transformation of 1 µmol of SOPC per minute.
The ß-elimination reactions were measured using a coupled assay with lactate dehydrogenase and NADH measured at 340 nm ( = 6.22x103 l/mol.cm), as described by Morino and Snell (1970) for tryptophanase. Reaction mixtures contained 50 mM potassium phosphate, pH 8.0, 50 µM pyridoxal-P, 0.2 mM NADH, 0.02 mg of lactate dehydrogenase and various amounts of amino acid substrate in a total volume of 0.6 ml, at 25°C. The reaction was initiated by the addition of enzyme solution. Determination of the kinetic parameters for SOPC was performed at 25°C in 50 mM potassium phosphate, pH 8.0, 5 mM 2-mercaptoethanol and 50 µM pyridoxal-P, with varying amounts of SOPC and appropriate dilutions of wild-type and mutant TPL.
The inhibitory effects of amino acids on the mutant TPL were determined using SOPC as substrate as described above.
Protein determination
Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. The concentration of purified TPL was determined from the absorbance at 278 nm (E1% = 8.37) (Muro et al., 1978), assuming a subunit molecular mass of 51.4 kDa (Antson et al., 1993
).
Isotope exchange experiments
The isotope exchange reactions with L-phenylalanine and L-methionine were carried out in 50 mM potassium phosphate buffer in the presence of 0.1 mM pyridoxal-P in a total volume of 4 ml. The pH of the solution measured potentiometrically with a glass electrode was equal to 8.2, which corresponds to p2H = 8.6 (Blesoe and Long, 1960). The concentration of L-phenylalanine was 28.6 mM, 6.7 mg of [N185A] TPL were added and aliquots of reaction mixture were withdrawn after 1, 2, 3, 5 and 7 h and heated at 90°C for 5 min to stop the reaction and then analyzed by 1H NMR. In the reaction with L-methionine, the concentration of the latter was 132 mM, 3.34 mg of [N185A] TPL were added and aliquots were withdrawn after 5, 8, 26, 35 and 51 h and treated as above. The ratio of deuterated to non-deuterated products was calculated from the relative integral intensities of the -proton and ß-CH2 group signals, the latter being used as an internal standard.
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 usung a short desalting column (PD-10, Pharmacia) equilibrated with 50 mM potassium phosphate, pH 8.6, and the activity of the preparation was measured. No loss of activity was observed. Rapid-scanning stopped-flow kinetic data were obtained with an RSM instrument from OLIS. 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 50 mM potassium phosphate, pH 8.6, 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 LMFT or SIFIT programs provided by OLIS. Validity of the fitting was evaluated by standard deviation or the DurbinWatson parameter. Typically, rate constants were estimated to a standard deviation of <5%.
Data analysis
Steady-state kinetic values of kcat and Km were obtained by fitting the data (initial velocity versus substrate concentration) to the MichaelisMenten equation using a hyperbolic regression program (Hyper).
Calculations of inhibition constants (Ki) were performed by using the FORTRAN programs of Cleland (1979) adapted to run on IBM-compatible personal computers. The data were fitted to the equation
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To calculate kinetic isotope effects the same programs were used, the data being fitted to the equation
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The concentration dependences of the absorbance amplitudes were fitted to an equation formally analogous to the MichaelisMenten equation and the respective affinity values, denoted Ks, were obtained.
Pre-steady-state kinetic apparent rate constants from stopped-flow experiments were fitted to Eqn (5) (Strickland et al., 1975
), using Enzfitter (Elsievier), where kf and kr are the forward and reverse rate constants, respectively.
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In the isotope exchange experiments the dependence of product concentration [P] on time t with an allowance for enzyme inhibition by the product is given by the equation
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where Kp is the affinity of the enzyme for the deuterated product and [S]0 is the initial concentration of the substrate.
The traditional treatment of data (Foster and Niemann, 1953) was applied to obtain the initial rates from the time course of the product concentration.
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Results |
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Concentration dependences of initial rates for reactions of [N185A] TPL with L-tyrosine, 3-fluoro-L-tyrosine, S-methyl-L-cysteine, S-ethyl-L-cysteine, S-benzyl-L-cysteine, SOPC and ß-chloro-L-alanine obeyed the MichaelisMenten equation and the main kinetic parameters for these reactions are presented in Table I.
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3-Fluoro-L-tyrosine.
The interaction of [N185A] TPL with 3-fluoro-L-tyrosine was characterized by a decrease of internal aldimine absorbance ( = 420 nm) and the appearance of new species absorbing at 345 and 500 nm (Figure 1
). The first absorbance, which appeared more rapidly, may be ascribed to a gem-diamine structure and the second one evidently belongs to the quinonoid intermediate. It was possible to follow the time course for gem-diamine formation, which was satisfactorily described by a single exponential process. The dependence of kobs on concentration of 3-fluoro-L-tyrosine (Figure 2
) was fitted by Equation 5,
thus the reaction pattern obeys the kinetic Scheme 2 and the respective kinetic parameters for gem-diamine formation are presented in Table V
. The kinetic curves for quinonoid formation were fitted by one exponential process. The rate constants (kobs) did not depend on the substrate concentration. The average value was estimated at 50.9 ± 2.6 s1. The concentration dependence of the amplitude of absorbance at 500 nm was well described by an equation formally analogous to the MichaelisMenten equation. The value of Ks = 0.9 ± 0.08 mM was determined from these data and is in good agreement with the value of Km = 0.78 mM found in steady-state experiments.
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Discussion |
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The mutation effect on the binding of TPL with substrates and inhibitors
To evaluate the role of Asn185 on the affinity of the enzyme for amino acids we examined the relationship between the structure of various amino acids and the efficiency of their binding to the mutant TPL as reversible competitive inhibitors. For wild-type TPL it has been established (Faleev et al. 1988) that for a number of amino acids bearing non-branched substituents without functional groups, the hydrophobicity of the side chain is the main factor controlling Ki. Amino acids that contain nucleophilic side chains exhibit enhanced affinities for the enzyme. It was supposed that these nucleophilic substituents interact with an electrophilic group in the active site. Evidence was presented by Mouratou et al. (1999) that Arg100 occupies a suitable position to perform such an interaction. In the present work we determined Ki values for characteristic representatives of both groups of amino acids for the mutant TPL N185A. These data are presented in Table III
and Figure 6
. It is evident (Figure 6
) that for the non-branched amino acids there is a linear relationship with a slope of ~1 between the values of RTlnKi for the mutant and wild-type TPLs:
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Thus, for the non-branched amino acid inhibitors the hydrophobicity of the side chain remains the main parameter controlling Ki when Asn185 is replaced by Ala. The absolute affinity values are reduced for the mutant by an average 0.69 ± 0.35 kcal/mol. On the other hand, it may be seen (Figure 6) that for most amino acids belonging to the second group of inhibitors, the affinities for the mutant enzyme are decreased much more and the respective points show large negative deviations from the straight line. For most of the tested substrates the affinities are also decreased considerably more (from 1.55 to 0.9 kcal/mol) on comparing the mutant with the wild TPL, as can be calculated from the Km values presented in Table I
.
It is noteworthy that Kis determined from steady-state kinetic experiments are effective values which may be described by Equation 7. Thus, to explain the observed changes in the overall affinities of various amino acids, the effects of N185A substitution on Kd and Kq should be taken into account. Toward this end we compared the interaction of TPL N185A with L-methionine and L-phenylalanine. The respective data were obtained by the stopped-flow technique. In both cases the reaction pattern followed the general Scheme 2, which allowed separate evaluation of the respective values of Kd and Kq. In Table IV
these parameters are compared with the analogous data for wild-type TPL. For both amino acids the effect of the mutation on the value of Kd is comparatively small, whereas Kq decreases considerably for the mutant TPL. This effect is more pronounced for L-methionine than for L-phenylalanine; however, the following additional consideration should be taken into account to explain the especially strong influence of the mutation on the affinity for L-methionine. For L-methionine Kq is much more than 1 and consequently the decrease in the value of Kq for the mutant TPL is reflected largely in the value of Ki, whereas in the case of L-phenylalanine, when Kq is comparable to 1, this effect is considerably dampened.
It is interesting that complexes of wild-type TPL with L-homoserine and L-aspartic acid, like the complex with L-methionine, exhibit intense absorbance at 500 nm. Thus, one may assume that negative deviations from the straight line in Figure 6 for all the three amino acids result from destabilization of the quinonoid intermediate for the mutant TPL and not from particularities of binding when the side chain interacts with Arg 100. This assumption is substantiated by the fact that the value for L-glutamic acid does not deviate significantly from the straight line. L-Glutamic acid belongs to the second group of amino acid inhibitors, but its complex with wild-type TPL does not display significant absorbance at 500 nm, so the amount of quinonoid formed is negligible. Together these results indicate that replacement of Asn185 residue with Ala results in a significant destabilization of quinonoid intermediates formed with amino acid substrates and inhibitors. This effect is stronger than destabilization of respective external aldimines. Evidently, the hydrogen bond between Asn185 and oxygen atom at C-3 of the cofactor, which is formed in the external aldimine, is maintained in the quinonoid intermediate. In this case, stabilization brought about by this bond should increase on going from the external aldimine to the quinonoid intermediate. This is because deprotonation of C-
atom leads to an increase in the total electron density in the
-electron system incorporating the 3'-O atom of pyridoxal-P and consequently the energy of its hydrogen bond with Asn185 should also increase.
The mutation effect and the distribution of intermediates
The interaction of TPL with any amino acid is, in general, a multi-stage process including a number of equilibria (Scheme 1). Thus, taking into account the relative destabilization of the external aldimine and quinonoid, it was reasonable to expect that other forms of enzymesubstrate complexes would be present in greater amounts in the resulting distributition of intermediates with the mutant TPL. This was demonstrated by spectral studies of complexes formed by TPL N185A with two aromatic amino acids, 3-fluoro-L-tyrosine and L-phenylalanine, the former being a substrate and the latter a reversible competitive inhibitor. Along with the absorbance peak at 500 nm, which is characteristic for the quinonoid intermediate, intense peaks were observed at 340350 nm in both cases (Figure 1). Structures absorbing at these wavelengths have been observed previously among the complexes of the wild-type TPL with 2,3-, 2,5- and 2,6-difluorotyrosine (Phillips et al., 1997
). Transient structures displaying the same spectral properties were also formed upon the interaction of tryptophanase with substrate analogues (Roy et al., 1988
). The absorbance at 340350 nm may be ascribed either to a gem-diamine (structure II, Scheme 1) or an
-aminoacrylate (structure VI, Scheme 1). The latter cannot be formed in the case of L-phenylalanine for which elimination of the side group is impossible for chemical reasons, although in the reaction with 3-fluorotyrosine
-aminoacrylate might, in principle, be formed. In reactions with both amino acids species absorbing at 340350 nm appeared more rapidly than did quinonoid. Taking into account that quinonoid is an intermediate in the pathway to
-aminoacrylate, we believe that in both cases the absorbance at 340350 nm should be ascribed to the gem-diamine. For the reaction with 3-fluoro-L-tyrosine we were able to study the kinetics of gem-diamine formation by the stopped-flow method. The reaction obeyed the formal Scheme 2. The concentration dependence of kobs is given in Figure 2
and the respective kinetic parameters are presented in Table V
. The observed value of Kd probably reflects non-covalent substrate binding to form a `Michaelis complex'. The rate constants kf and kr describe the velocities of addition of the substrate amino group to the double bond of the external aldimine to form the gem-diamine and the reverse reaction of dissociation of the gem-diamine, respectively.
The -proton lability in TPL complexes with amino acids and the rates of
-proton isotope exchange catalyzed by TPL in 2H2O
Both wild-type and [N185A] TPLs catalyze isotope exchange of the -protons of some amino acids for deuterium in 2H2O. We compared the observed rates of exchange with the kinetic parameters for the quinonoid formation for reactions of wild-type and N185A TPLs with L-phenylalanine and L-methionine. These data are presented in Table IV
. Interestingly, for reactions of the mutant enzyme with both inhibitors the decrease in equilibrium constant for deprotonation (Kq = kf/kr) results solely from acceleration of the reprotonation reaction. Deprotonation rates do not decrease, as might be expected, but actually increase and in the case of L-methionine increase considerably (by a factor of 5.7). This may be explained by a conformational change occurring in the mutant enzyme, which ensures a more favorable relative orientation between the
-proton in the external aldimine and the basic group accepting this proton.
For L-methionine the exchange rates in both cases are comparable with the quinonoid reprotonation rates (for the wild-type enzyme kexch for unknown reasons is even somewhat higher than kr), while deprotonation of the external aldimine proceeds much faster. For N185A TPL the exchange rate for L-methionine is accelerated by a factor of 7.4 as compared with wild-type, which is in accord with the increase in lability of the -proton. On the other hand, for L-phenylalanine the exchange rates with the wild-type enzyme are much slower than the observed values of kf and kr. Replacement of Asn185 with Ala is accompanied by a considerable increase in lability of the
-proton (kf and kr for L-phenylalanine are increased), whereas the exchange rate remains almost unchanged. These data may be interpreted as evidence that the internal return of
-proton after its abstraction proceeds faster than its exchange with the solvent 2H2O. Evidently, the internal return becomes especially important for the N185A TPL. This assumption deserves some comment in connection with the question of the basic group responsible for the
-proton abstraction. According to the X-ray data (Antson et al., 1993
) Lys 257 seems a probable candidate for this role. In 2H2O after the
-proton abstraction the amino group should bear two deuterium and one hydrogen atoms and, consequently, statistical factors should favor the transfer of a deuteron, but not the proton to the quinonoid intermediate. Thus, to ensure the observed extent of internal return, free rotation of the
-amino group should be restricted through hydrogen bond(s) to the protein. At the same time, any hydrogen bond of a considerable strength in which a non-protonated amino group takes part should involve the lone electron pair on the nitrogen. The latter, however, must be free to accept the substrate's
-proton. It seems likely that the hydrogen bond is formed in a concerted way, the proton transfer being accompanied by development of a positive charge on the nitrogen.
Systematic studies of the mechanism of C-proton exchange in reactions of TPL with a number of amino acids are now in progress.
The effect of the mutation on the reactivity of the substrates and reaction mechanism
Kinetic data characterizing the reactions of TPL N185A with various substrates are presented in Table I in comparison with analogous data for the wild-type enzyme. In general, higher Km values are found for the mutant TPL than for the wild-type enzyme with the same substrates. This is to be expected taking into account the destabilization of the quinonoid intermediate and, possibly, the external aldimine, brought about by the mutation.
The influence of this mutation on the values of kcat for the substrates bearing good leaving groups at the ß-position may be explained when two considerations are taken into account. First, the destabilization of the quinonoid intermediate should lead to a decrease in its content under steady-state conditions, which should retard the reaction. Second, removal of the hydrogen bond between Asn185 and oxygen at the 3'-position of the cofactor should increase the negative charge of the quinonoid intermediate and, consequently, the quinonid in the mutant enzyme should be more reactive for the elimination of the leaving group. The experimental results show that for the reactions of S-ethylcysteine, S-methylcysteine and SOPC the two effects to a considerable degree counterbalance each other, whereas in the case of ß-chloroalanine the effect leading to acceleration is predominant. The results are qualitatively different for reactions with the natural substrate, L-tyrosine, and its closest analogue, 3-fluoro-L-tyrosine. For these substrates the mutation results in a significant decrease in the kcat values by factors of 42 and 59, respectively. According to the generally accepted mechanism of TPL reactions with suitable tyrosine-type substrates (Scheme 1), the main chemical transformations proceed in the course of the three principal stages: (a) quinonoid formation as the result of -proton abstraction in the internal aldimine; (b) tautomerization of the phenol moiety to convert it into a good leaving group; and (c) ß-elimination of the leaving group with regeneration of its aromaticity. These stages are sensitive to different kinetic isotope effects: stage (a) to
-deuteration of the substrate (
-KIE), stage (b) to solvent isotope effect (SKIE) on going from water to 2H2O and stage (c) to ß-deuteration of the substrate (ß-KIE). In our earlier work (Faleev et al., 1996
) we investigated KIEs that influence different elementary stages for the reactions of Erwinia herbicola TPL with L-tyrosine and 3-fluoro-L-tyrosine. We found that stages (a) and (b) should be at equilibrium while the highest maximum on the free-energy profile corresponds to stage (c).
In the framework of the kinetic Scheme 3, taking account of the three principal stages, the main kinetic parameters are given by Equations 8 and 9.
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For the suggested mechanism (Faleev et al, 1996) it should be accepted that (i) krt > ke and (ii) krt > kt, so, to a first approximation kcat can be described by Equation 10.
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For the mutant enzyme the decrease in quinonoid content under steady-state conditions described by the factor of kf/(kf + kr) should be to a considerable degree compensated by the increase in ke value, both effects being the consequence of the destabilization of the quinonoid intermediate, as is observed for substrates bearing good leaving groups. Thus, the observed considerable decrease in rates for L-tyrosine and 3-fluoro-L-tyrosine might be associated with stage (b). In Table II the values of various KIEs are presented for reactions of the mutant and wild-type TPLs with L-tyrosine and 3-fluoro-L-tyrosine.
The values of -KIEs for the reaction of [N185A] TPL with 3-fluoro-L-tyrosine are comparable to the analogous data for wild-type TPL. In the case of the mutant enzyme the
-KIE on the kcat/Km value is much greater than the respective KIE on kcat, which may be interpreted as evidence for retardation of some stage(s) occurring after
-proton abstraction (Kiik and Phillips, 1988). The solvent KIE on the reaction of L-tyrosine with TPL N185A is even lower than that for wild-type TPL, hence the unfavorable effect of the mutation on the tautomerization stage evidently does not make this stage rate-limiting. The comparatively high ß-KIE observed for the reaction of N185A TPL with 3-fluoro-L-tyrosine allows us to conclude that the ß-elimination stage remains formally rate-limiting and the principal kinetic mechanism for reactions of TPL with adequate substrates is not changed by replacing Asn185 with Ala. Consequently, the observed decrease in kcat for L-tyrosine and 3-fluoro-L-tyrosine should result from an unfavorable effect of the mutation on the tautomerization equilibrium (KT = kt/krt), which means that Asn185 is involved in an additional stabilization (by 2.22.4 kcal/mol) of keto quinonoid intermediate (structure V, Scheme 1) in the wild-type enzyme. This may be due to participation of Asn185 in a system of hydrogen bonds in the active site, although the exact origin of this effect at present is not evident. According to X-ray data for the complex of the enzyme with N-(5'-phosphopyridoxyl)-L-tyrosine, which mimics the external aldimine (Pletnev et al., 1997
), the carbonyl oxygen of Asn185 is 3.54 Å from the guanidinium group of Arg404 which binds the substrate's carboxylic group (Sundararajiu et al., 1997), but the distances between Asn185 or Arg404 and other residues in the active site are too high to ensure a significant interaction and it is not clear in which way a network leading to a stabilization of the keto quinonoid might be formed, Thus, X-ray studies on structures modeling the keto quinonoid are necessary to arrive at a decisive conclusion.
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Acknowledgments |
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Notes |
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References |
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Chen,H., Demidkina,T.V. and Phillips,R.S. (1995a) Biochemistry, 34, 1227612283.[ISI][Medline]
Chen,H., Gollnick,P. and Phillips,R.S. (1995b) Eur. J. Biochem., 229, 540549.[Abstract]
Cleland,W.W. (1979) Methods Enzymol., 63, 103138.[Medline]
Demidkina,T.V., Myagkikh,I.V. and Azayev,A.V. (1987) Eur. J. Biochem., 170, 311316.[Abstract]
Enei,H., Matsui,H., Yamashita,K., Okumura,S. and Yamada,H. (1972) Agric. Biol. Chem., 36, 18611868.[ISI]
Faleev,N., Ruvinov,S.B., Demidkina,T.V., Myagkikh,I.V., Gololobov,M.Y., Bakhmutov,V.I. and Belikov,V.M. (1988) Eur. J. Biochem., 177, 395401.[Abstract]
Faleev,N.G., Spirina,S.N., Demidkina,T.V. and Phillips,R.S. (1996) J. Chem. Soc., Perkin Trans. 2, 20012004.
Foster,R.J. and Niemann,C. (1953) Proc. Natl Acad. Sci USA, 39, 999.[ISI]
Glasoe,P.V. and Long,F.A. (1960) J. Phys. Chem., 64, 188194.[ISI]
Jansonius,J.N. (1998) Curr. Opin. Struct. Biol., 8, 759769.[ISI][Medline]
Kiick,D.M. and Phillips.R.S. (1988) Biochemistry27, 73337338.[ISI][Medline]
Kumagai,H., Matsui,H., Ohkishi,H., Ogata,K., Yamada,H., Ueno,T. and Fukami,H. (1969) Biochem. Biophys. Res. Commun., 34, 266270.[ISI][Medline]
Kumagai,H., Yamada,H., Matsui,H., Ohkishi,H. and Ogata,K. (1970) J. Biol. Chem., 245, 17671777.
Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.P. (1951) J. Biol. Chem., 193, 265.
Morino,Y. and Snell,E.E. (1970) Methods Enzymol., 17A, 439446.
Mouratou,B., Kasper,P., Gehring,H. and Christen,P. (1999) J. Biol. Chem., 247, 13201325.
Muro,T., Nakatani,H., Hiromi,K., Kumagi,H. and Yamada,H., (1978) J.Biochem., 84, 633640.[ISI][Medline]
Phillips,R.S. (1987) Arch. Biochem. Biophys., 256, 302310.[ISI][Medline]
Phillips,R.S., Ravichandran,K.and Von Tersch,R.L. (1989) Enzyme Microb.Technol., 11, 8083.
Phillips,R.S., Fletcher,J.G., Von Tersch,R.L. and Kirk,K.L. (1990) Arch. Biochem. Biophys., 276, 6569.[ISI][Medline]
Phillips,R.S., Von Tersch,R.L. and Secundo,F. (1997) Eur. J. Biochem., 244, 16581666.
Pletnev,S.V. et al. (1997) Kristallografiya, 42, 877888 (in Russian).
Roy,M., Miles,E.W., Phillips,R.S. and Dunn,M.F. (1988) Biochemistry, 27, 86618669.[ISI][Medline]
Sanger,F., Nicklen,S. and Coulsen,A.R. (1977) Proc. Natl Acad. Sci. USA, 74, 54635467.[Abstract]
Strickland,S., Palmer,G. and Massey,V. (1975) J. Biol. Chem., 250, 40484337.[ISI][Medline]
Sundararaju,B., Antson,A.A., Phillips,R.S., Demidkina,T.V., Barbolina,M.V., Gollnick,P., Dodson,G.G. and Wilson,K.S. (1997) Biochemistry, 36, 65026510.[ISI][Medline]
Taylor,J.,W., Ott,J. and Eckstein,F. (1985) Nucleic Acids Res., 13, 87658785.[Abstract]
Vieira,J. and Messing,J. (1987) Methods Enzymol., 153, 134.[Medline]
Ueno,T., Fukami,H., Ohkishi,H., Kumagai,H. and Yamada,H. (1970) Biochim. Biophys. Acta, 206, 476479.[ISI][Medline]
Yamada,H., Kumagai,H., Kashima,N., Torii,H., Enei,H. and Okumura,S. (1972) Biochem. Biophys. Res. Commun., 46, 370374.[ISI][Medline]
Yano,T., Mizuno,T. and Kagamiyama,H. (1993) Biochemistry, 32, 18101815.[ISI][Medline]
Received August 8, 1999; revised January 10, 2000; accepted January 19, 2000.