Thermodynamics and kinetic aspects involved in the enzymatic resolution of (R,S)-3-fluoroalanine in a coupled system of redox reactions catalyzed by dehydrogenases

Article abstract of DOI:10.1021/op060027o

Two systems of redox enzymatic reactions were tested, looking forward to the preparation of (S)-3-fluoroalanine, a potent antibiotic, by kinetic resolution of rac-3-fluoroalanine. This starting material was the main substrate for the deaminative oxidation reaction catalyzed by L-alanine dehydrogenase (L-AlaDH) in the presence of NAD⁺. One system was formed by coupling this reaction (main reaction) to the reduction of 3-fluoropyruvate (a cascade system) produced in the main reaction catalyzed by L-lactate dehydrogenase (L-LDH) in the presence of NADH, also formed in the main reaction. This system, that was able to achieve 92% of conversion, allows the accumulation of NH ₄⁺, one of the secondary products of the main reaction. The other coupled redox system involved the coupling to the L-AlaDH reaction to the aminative reduction reaction of α-ketoglutarate in the presence of NADH and NH₄⁺ (both side products of the main reaction) catalyzed by L-glutamate dehydrogenase (L-GluDH), that allows accumulation of 3-fluoropyruvate. With this system, the extent of the reaction in the coupled system was only 22%. This big difference in the efficiency of both systems was identified as being the result of a different potency of the products that accumulates in both systems, acting as inhibitors of L-AlaDH. It was demonstrated that 3-fluoropyruvate is a much stronger inhibitor of L-AlaDH than NH₄⁺. This fact, and not thermodynamic considerations, explains the results obtained with both systems.

Full text of DOI:10.1021/op060027o

Organic Process Research & Development 2006, 10, 673−677
Thermodynamics and Kinetic Aspects Involved in the Enzymatic Resolution of
(R,S)-3-Fluoroalanine in a Coupled System of Redox Reactions Catalyzed by
Dehydrogenases
Luciana P. B. Gonc¸alves, O. A. C. Antunes,* and Enrique G. Oestreicher*
Instituto de Qu´ımica, UFRJ, CT Bloco A 536B, Cidade UniVersita´ria, Rio de Janeiro RJ 21945-970, Brasil
Abstract:
Two systems of redox enzymatic reactions were tested, looking
forward to the preparation of (S)-3-fluoroalanine, a potent
antibiotic, by kinetic resolution of rac-3-fluoroalanine. This
starting material was the main substrate for the deaminative
oxidation reaction catalyzed by L-alanine dehydrogenase (L-
AlaDH) in the presence of NAD+. One system was formed by
coupling this reaction (main reaction) to the reduction of
3-fluoropyruvate (a cascade system) produced in the main
reaction catalyzed by L-lactate dehydrogenase (L-LDH) in the
Figure 1. Coupled enzymatic redox system for the simulta-
neous production of (S)-3-fluoroalanine (1a) and (R)-3-fluoro-
lactic acid (3) using rac-3-fluoroalanine (rac-1), (R)-3-fluoro-
alanine (1b), 3-fluoropyruvic acid (2). The regeneration reaction
was constituted by the enzymatic reduction of 2 catalyzed by
L-LDH in the presence of NADH both produced in the main
reaction (oxidative deamination of (R)-fluoroalanine).
presence of NADH, also formed in the main reaction. This
system, that was able to achieve 92% of conversion, allows the
accumulation of NH4+, one of the secondary products of the
main reaction. The other coupled redox system involved the
coupling to the
L
-AlaDH reaction to the aminative reduction
+
reaction of r-ketoglutarate in the presence of NADH and NH4
(both side products of the main reaction) catalyzed by
glutamate dehydrogenase ( -GluDH), that allows accumulation
On the other hand, (S)-3-fluoroalanine has been described
as a potent antibiotic of wide spectrum.3 Its mechanism of
action involves the irreversible inactivation of the typical
bacterial enzyme, alanine racemase,4 an enzyme involved
in the biosynthesis of the cell wall. This nonproteinogenic
amino acid also acts as an inhibitor of serine palmitoyl
transferase;5 in addition, it is a potential precursor of
fluoroamine compounds.6,7
Both fluorinated compounds can be produced simulta-
neously by using a coupled enzymatic system involving
dehydrogenases. However, an enzymatic system of this type
necessarily involves the regeneration of the redox active form
of the nicotinamide coenzyme, since it cannot be used in
stoichiometric concentration with the other substrate of the
reaction system.
Our group has been involved in the production of (S)-3-
fluoroalanine8 via kinetic resolution of rac-3-fluoroalanine
by deaminative oxidation catalyzed by L-AlaDH. (R)-3-
Fluorolactic acid was simultaneously produced via L-LDH-
catalyzed enantioselective reduction of 3-fluoropyruvate,
generated in the former reaction (Figure 1).
In the development of the process leading to a possible
industrial use, a more thermodynamically suitable redox
enzymatic system was tested. This coenzyme regeneration
L
-
L
of 3-fluoropyruvate. With this system, the extent of the reaction
in the coupled system was only 22%. This big difference in the
efficiency of both systems was identified as being the result of
a different potency of the products that accumulates in both
systems, acting as inhibitors of
L
-AlaDH. It was demonstrated
-AlaDH
that 3-fluoropyruvate is a much stronger inhibitor of
L
than NH4+. This fact, and not thermodynamic considerations,
explains the results obtained with both systems.
1. Introduction
Fluorinated chiral R-hydroxyacids, such as (R)-3-fluoro-
lactic acid, are considered to be highly versatile chiral
building blocks in asymmetric synthesis for the production
of several compounds of pharmacological interest, for
example, â-adrenergic blocking agents, such as (S)-propra-
nolol and (S)-moprolol, and other products of pharmaceutical
interest such as (S)-3-hydroxypyrrolidine-2-one which in its
open form, (S)-4-amino-2-hydroxybutanoic acid, is consid-
ered to be one of the most potent known inhibitors of the
neurotransmitter GABA and also shows anticancer activity.1,2
In addition, such a compound is the ideal precursor for either
(S)- or (R)-3-fluoroalanine, via triflate formation and one
inversion or chloride formation and two inversions.
(3) (a) Kollonitsch, J.; Barash, L.; Kahan, F. M.; Kropp, H. J. Am. Chem. Soc.
1988, 110, 2959. (b) Grabowski, E. J. J. Chirality 2005, 17, S249.
(4) Esaki, N.; Walsh, C. T. Biochemistry 1986, 25, 3261.
(5) Sandvig, K.; Barred, O.; van Helvoort, A.; van Meers G.; van Deurs, B.
Mol. Biol. Cell. 1996, 7, 1391-404.
* Corresponding authors. E.G.O. E-mail: enrique@iq.ufrj.br. Telephone: +55
21 25627366. Fax: +55 21 25627559. O.A.C.A. E-mail: octavio@iq.ufrj.br.
Telephone: +55 21 25627818. Fax: +55 21 25627559.
(1) Gonc¸alves, L. P. B.; Antunes, O. A. C.; Pinto, G. F.; Oestreicher, E. G. J.
Mol. Catal. B: Enzym. 1998, 4, 67.
(6) Schengrund, C. L.; Kovac, P. Carbohydr. Res. 1999, 319, 24.
(7) Hutton, C. A. Org. Lett. 1999, 1, 295.
(2) Bentley, J. M.; Wadsword, H. J.; Willis, C. L. J. Chem. Soc., Chem.
Commun. 1995, 231.
(8) Gonc¸alves, L. P. B.; Pinto, G. F.; Antunes, O. A. C.; Oestreicher, E. G.
Tetrahedron: Asymmetry 2000, 11, 1465.
10.1021/op060027o CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/06/2006
Vol. 10, No. 3, 2006 / Organic Process Research & Development
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Reaction mixtures contained in a total volume of 1 mL:
50 mM sodium phosphate buffer, pH 7.9, 20 mM rac-3-
fluoroalanine, and 0.25 mM NAD+. Reaction was started
by adding 20 µL of an enzyme solution containing in a
volume of 1.0 mL: 130 µg (protein corresponding to 0.45
IU of a desalinized L-AlaDH enzyme preparation (obtained
by ultrafiltration with Centricon-10 concentrators (AMI-
CON), through five consecutive centrifugations at 5 °C for
1 h and 2000g and reconstitution of the original volume with
sodium phosphate buffer 10 mM, pH 7.7, and 50% (v/v) of
glycerol. The slopes of the recording lines were kept close
to 45° by varying the absorbance full scale and/or the time
full scale of the spectrophotometer. The printer curves
obtained were extrapolated to the time of enzyme addition,
and the tangents of the curves at this time were taken as
initial velocity. The initial velocity of the reaction was
calculated in terms of millimoles of NADH produced per
minute, using a molar absorption coefficient for NADH of
6220 M-1 cm-1.
Figure 2. Alternative coupled redox system for the enzymatic
production of (S)-3-fluoroalanine. The substrate for AlaDH was
rac-3-fluoroalanine (rac-1) which deaminated oxidatively the
(R)-enantiomer of the racemic amino acid (1b) producing
3-fluoropyruvic acid (2) and leaving unreacted (S)-3-fluoroala-
nine (1a), r-ketoglutarate (3), (S)-glutamate (4).
system was constituted by L-GluDH and R-ketoglutarate
(Figure 2). This enzymatic system has the advantage over
the former one (L-LDH and 3-fluoropyruvate) that the
chemical reaction involved has a greater thermodynamic
driving force (∆G′o) than the reduction of 3-fluoropyruvate
to 3-fluorolactate (supposing E′o equals that of the half-
reaction pyruvate/lactate).
In the present paper the performances of both recycling
systems were compared, and the higher efficiency of the
3-fluoropyruvate/3-fluorolactate recycling system was at-
tributed to a kinetic effect, strong inhibition of L-AlaDH by
3-fluropyruvate, a product accumulating just with the R-ke-
toglutarate/glutamate system. This inhibition of the enzyme
catalyzing the main reaction, deaminative oxidation of (L)-
3-fluoroalanine, by a reaction product that it is not eliminated
by the recycling reaction is a much more important factor
than the higher thermodynamic driving force supplied by
the other coenzyme recycling system. This, therefore,
explains the success of the cascade reaction employed.
2.2. Inibition of AlaDH by 3-Fluoropyruvate and
+
NH4 . Assays of AlaDH activity under conditions of initial
rate were performed as described in section 2.1 using a
constant concentration of both rac-3-fluoroalanine (4.5 mM)
and of NAD+ (0.25 mM). Each product inhibitor (3-
+
fluoropyruvate and NH4 ) was added to the respective
reaction mixture in concentrations ranging from zero to 5.0
mM, as indicated in the legend of Figure 4.
2.3. Coupled Enzymatic Redox Systems for NAD+
Regeneration. Reaction was performed in a jacketed batch
reactor of 30 mL, continuously stirred. The reaction medium
contained, in a total volume of 12.5 mL, 0.05 mM of rac-
3-fluoroalanine, sodium phosphate buffer, pH 7.9, 20 mM
rac-3-fluoroalanine, 90 IU of L-AlaDH, 1000 IU of L-LDH
(or 50 mM R-ketoglutarate and 950 IU of L-GluDH).
Reactions were started by adding 0.25 mM NAD+. Reaction
mixtures were maintained under continuous magnetic stirring,
and the temperature was maintained at 25 °C by circulating
water through the jacket of the reactor with a thermocircu-
lating bath.
2. Experimental Section
Bacillus subtilis L-alanine dehydrogenase (L-AlaDH) (L-
alanine:NAD+ oxidoreductase, EC 1.4.1.1), suspension in 2.4
M (NH4)2SO4, pH 7.0, L-lactate dehydrogenase (L-LDH) from
rabbit muscle (L-(+)-lactate:NAD+ oxidoreductase, EC
1.1.1.27), solution in 50% glycerol containing 10 mM
potassium phosphate buffer, pH 7.5, L-glutamate dehydro-
genase.(L-GluDH) (L-glutamate:NAD(P)+ oxidoreductase,
EC 1.4.1.3), lyophilized, NAD+ (grade III), NADH (grade
III), bovine serum albumin (fraction V), sodium pyruvate,
sodium 3-fluoropyruvate, and Dowex 50W-X8 cation-
exchange resin were obtained from Sigma. R-Ketoglutarate,
NaBH4, and the Diazald kit (for CH2N2 generation were
purchase from Aldrich Chemical. All other chemicals were
of analytical grade and were obtained from Merck Darmstad.
2.1. Assay of L-AlaDH Activity. L-AlaDH activity
(measured in the direction of rac-3-fluoroalanine deaminative
oxidation) was assayed by following NADH absorption at
340 nm at pH 7.9 and 25 °C. All assays were carried out in
quartz cuvettes with a 1-cm light path using a Beckman DU
70 spectrophotometer equipped with a dot-matrix printer.
The temperature of the cell holder was kept at 25 °C by
forced circulating water.
The extent of reaction was determined by removing
aliquots of the reaction medium (10-50 µL) at different
times. These aliquots were immediately diluted with 0.05
M sodium phosphate buffer, pH 7.9, and heated at 100 °C
for 5 min to inactivate the enzymes. The concentration of
+
NH4 (reaction system represented by Figure 1) or of
3-fluoropyruvate (reaction system shown in Figure 2) present
in the corresponding samples was determined by “end-point”
assays. NH4+ was assayed in a solution of 0.2 mM NADH,
0.5 mM of R-ketoglutarate, and 1 mg of L-GluDH in a total
volume of 2.0 mL at 25 °C. The concentration of 3-fluoro-
pyruvate that accumulated in the reaction medium was
determined in the presence of 0.25 mM NADH and 14 IU
of L-LDH in a total volume of 2.0 mL at 25 °C.
2.4. Determination of Enantiomeric Excesses. The
enantiomeric excesses of the target products were determined
by chiral HPLC which was performed with an ISCO 2350
chromatograph equipped with a Nucleosil Chiral-1 column
(Macherey-Nagel, ET 250/8/4) by using 1 mM CuSO4, pH
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Vol. 10, No. 3, 2006 / Organic Process Research & Development

Table 1. Standard free energy changes resulting for the coupling of desaminative oxidation enzymatic reaction of
rac-3-fluoroalanine with the two coenzyme regeneration enzymatic reactions
redox reaction
Eo′ (v)
∆Go′ (cal/mol)
alanine + H2O + NAD+ T pyruvate + NH4+ + NADHa
pyruvate + NADH T lactate + NAD+ b
-0.19
0.13
0.18
2760a
466b
bR-ketoglutarate + NH4+ + NADH T glutamate + H2O + NAD+ b
a Considering reactions a and b and ∆Go′ ) -nF∆Eo′ where n ) 2, F ) 23 000 (cal/absolute volt equivalent) and ∆Eo′ the difference of standard redox potentials
between the half reactions considered. b Considering reactions a and c, ∆Go′ ) -nF∆Eo′ and the values of n, F, and the corresponding ∆Eo′ value.
reactions for the preparation of enantiopure or highly
enantioenriched chiral compounds. Reduction reactions are
important in asymmetric synthesis and oxidation reactions
and in kinetic resolutions of racemates.
Our research group has used both types of applications
with good results.1,8,9,12-17 However, the use of dehydroge-
nases in preparative applications requires effective processes
of coenzyme regeneration since these cofactors are too
expensive to be used as stoichiometric reagents. Moreover,
since oxidation (or oxidative deamination) reactions are
thermodynamically unfavorable, coenzyme regeneration by
coupling to these reactions (strongly exergonic reduction
reactions) drives the main reaction in the desired direction,
making the whole system of coupled reactions to behave
exergonically, allowing the main or desired reaction to
proceed with high yields. On the other hand, with reduction
(or aminative reduction) reactions, coupling of an oxidation
coenzyme regeneration reaction allows the kinetic resolution
of racemate of oxidized compounds.1,8
Figure 3. Time-course curves of the coupled enzymatic systems
for the production of (S)-3-fluoroalanine in a laboratory
preparative scale (150 mg). The progress of reactions was
followed by chiral HPLC determination of (S)-3-fluoroalanine
as described in section 2.3.
Two coenzyme regeneration systems were envisaged to
allow the production of (S)-3-fluoroalanine. The first one,
shown in Figure 1, allowed the production of two chiral
compounds, our target product ((S)-3-fluoroalanine) (1a), a
potent antibiotic agent (3, 8-9), and (R)-3-fluorolactic acid
(3), a versatile chiral building block (1,12). The (S)-
enantiomer of the nonproteinogenic amino acid is obtained
by deaminative oxidation of the (R)-enantiomer (1b) of rac-1
4.6, as eluent at a flow rate of 1 mL/min, 23 °C, and UV
detection at 235 nm. Chemically synthesized rac-3-fluoro-
lactic acid and rac-3-fluoroalanine were used as standards.
The latter was obtained by reductive amination of 3-fluo-
ropyruvate. In this procedure 3-fluoropyruvate (3.5 mmol)
was treated with NABH4 (5.5 equiv) in the presence of NH4-
OH (68 mmol) as described in the literature.10 This product
as well as the target ones was isolated, purified, crystallized,
and identified as described previously by us.1,8,9
2.5. Determination of Protein Concentration. Protein
concentration was determined by using a method suitable
for the detection of low protein contents.11 Bovine serum
albumin (Fraction V) was used as the standard.
+
to produce NH4 , 3-fluoropyruvate (2), and NADH. The
latter two products of the main reaction are used by L-LDH
to produce (R)-3-fluorolactic acid (3) and NAD+. If the
velocity of the coenzyme recycling reaction is not rate
+
limiting of the whole process, just NH4 and (R)-3-fluoro-
alanine will accumulate in the reaction medium, and NAD+
will be continuously recycled until all 1b has been consumed.
Of course this picture will only be observed if the coenzyme-
recycling reaction has enough driving force (∆Go′) to direct
the system in this direction.
3. Results and Discussion
Oxidoreductions, especially the reduction of carbonyl
compounds, the aminative reduction of R-ketoacids, the
oxidation of hydroxyl groups, and the oxidative deamination
of rac-R-amino acids catalyzed by stereoselective NAD(P)+-
dependent dehydrogenases, are very important organic
(12) Menezes, R. R.; Santos, A. S.; Oestreicher, E. G.; Pinto, G. F. Biotechnol.
Tech. 1998, 12, 35.
(13) Gonc¸alves, L. P. B.; Antunes, O. A. C.; Pinto, G. F.; Oestreicher, E. G.
Tetrahedron: Asymmetry 1996, 5, 1237.
(14) Machado, G. D. C.; Gomes, M., Jr.; Antunes, O. A. C.; Oestreicher, E. G.
Proc. Biochem. 2005, 40, 3186.
(15) Arcuri, M. B.; Sabino, S. J.; Antunes, O. A. C.; Oestreicher, E. G. J.
Fluorine Chem. 2003, 121, 55.
(16) Arcuri, M. B.; Antunes, O. A. C.; Machado, S. P.; Almeida, C. H. F.;
Oestreicher, E. G. Amino Acids 2004, 27, 69.
(17) Machado, G. D.; Paiva, L. M. C.; Pinto, G. F.; Oestreicher, E. G. Enzyme
Microb. Technol. 2001, 28, 322.
(9) Gonc¸alves, L. P. B.; Antunes, O. A. C.; Pinto, G. F.; Oestreicher, E. G. J.
Fluorine Chem. 2003, 124, 219.
(10) Dolling, U. H.; Douglas, A. W.; Grabowski, E. J. J.; Schonewaldt, E. F.;
Sohar, P.; Sletzinger, M. J. Org. Chem. 1978, 43, 1634.
(11) Hartree, E. F. Anal. Biochem. 1972, 48, 422.
Vol. 10, No. 3, 2006 / Organic Process Research & Development
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Table 2. Experimental data used to plot Figure 3
Table 3. Experimental dataa of the inhibition of L-AlaDH by
3-fluoropyruvate and NH4
+
conversion (%)
residual activity (mM/min)
time (h)
L-LDHb
GluDHc
+
inhibitor concentration (mM)
3-fluoropyruvate
NH4
0
2
3
0
18
25
39
86
92
92
0
22
21.8
22
21.5
22
0
0.142
0.028
0.018
0.010
0.007
0.005
0.002
0.142
0.136
0.132
0.123
0.115
0.107
0.075
0.3
0.5
1.0
1.5
2.0
5.0
5
18
23
42
22
a The progress of reaction was followed by chiral HPLC determination of
(S)-3-fluoroalanine as described in section 2.3. b Coupled enzymatic system
formed with the L-LDH coenzyme recycling reaction (See Figure 1). c Coupled
enzymatic system formed with the GluDH/R-ketoglutarate NAD+ recycling
reaction (See Figure 2).
a These experimental data were used to plot Figure 4. Experimental details
are given in the caption of Figure 4 and in the Experimental Section.
Figure 5. Active site of L-AlaDH showing the interactions of
the Shiff base with the amino acid residues.
of unreacted (S)-3-fluoroalanine, our target product, will be
a direct function of the degree of conversion which, in turn,
will depend on the resulting driving force of the whole
system which is determined by the ∆Go′ of the coenzyme
recycling reaction.
However ∆Go′ is not the unique thermodynamic factor
involved since, in the first redox system (Figure 1 and Table
1), both NADH and pyruvate, two products of the main
reaction, are consumed by the recycling reaction, thus
increasing the driving force of the latter redox reaction. The
same picture happens with the recycling reaction shown in
Figure 2, where the products of the main reaction, NADH
+
Figure 4. Inhibition of AlaDH by 3-fluoropyruvate and NH4
.
Experimental conditions for the measurement of initial rate of
AlaDH-catalyzed reaction of oxidative deamination of rac-3-
fluoroalanine were given in section 2.1. Experimental details
for the inhibition of AlaDH by products of the main reaction
+
remaining with the L-LDH coenzyme recycling system (NH4
)
and the GluDH/r-ketoglutarate NAD+ recycling system, re-
+
and NH4 , are also removed by this recycling reaction. An
spectively, were given in section 2.2. The concentration of each
+
product inhibitor, (9) NH4 and (b) 3-fluoropyruvate, varied
extra effect of the removal of these products of the main
reaction is the elimination or removal of an inhibitor since
it is a well-known fact that in enzymic reactions, the products
formed are inhibitors of the reaction. Then, associated with
the thermodynamic effect exists a very important kinetic
effect that can influence the extent of the process.
from zero to 5.0 mM.
The other coenzyme regeneration system is shown in
Figure 2. In this system NAD+ produced in the main reaction
is regenerated by coupling to the deaminative oxidation
reaction of (R)-3-fluoroalanine catalyzed by L-AlaDH, the
aminative reduction of R-ketoglutarate is catalyzed by
Figure 3 shows the progress curve of the desaminative
oxidation reaction of rac-3-fluoroalanine with both recycling
reactions. With L-LDH as recycling enzyme, the system
reached 92% of conversion, whereas with the L-GluDH/R-
ketoglutarate system, the extent of the main reaction was
only of 22% (Figure 3) although this latter redox-coupled
system is thermodynamically more suitable than the former
one (Table 1). In Table 2 are displayed the experimental data
corresponding to these time-course curves. The enantiomeric
excess of (S)-3-fluoroalanine determined by chiral HPLC as
described in section 2.4 was of 86%. Since as shown in
Figure 1 with L-LDH as recycling enzyme, the only product
+
L-GluDH in the presence of NH4 , and NADH is produced
in the main reaction. This system is also able to produce
two chiral compounds (Figure 1), (S)-3-fluoroalanine and
(S)-glutamate.19 This latter system must be considered since,
as shown in Table 1, it has a much smaller positive ∆Go′
than the former one. This is an important issue to be
considered since L-AlaDH being absolutely stereospecific for
the (L or S)-enantiomer of alanine18 and also for the (L or
R)-enantiomer of 3-fluoroalanine,8,9 the enantiomeric excess
(18) Grimshaw, C. E.; Cleland, W. W. Biochemistry 1981, 20, 5650.
(19) O¨ dman, P.; Wellborn, W. B.; Bommarius, A. S. Tetrahedron: Asymmetry
2004, 15, 2933.
+
of the main reaction that accumulates is NH4 , whereas with
L-GluDH and R-ketoglutarate an accumulation of 3-fluoro-
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tion of each product inhibitor are given in Table 3. This
experiment then clearly indicated that the main reason for
the L-LDH coenzyme recycling reaction, although having a
lower thermodynamic driving force than the GluDH/R-
ketoglutarate system, is more efficient due to kinetic reasons
rather than to thermodynamic ones.
On the basis of the active site of a model L-AlaDH20 it is
reasonable to suppose that the intermediate, the Shiff base
complex, should interact with different amino acid residues,
such as a tyrosine residue (via π-stacking), that protects one
face from the hydride attack, a glutamic acid residue and an
arginine residue, as ionic pair interactions (Figure 5).
Therefore, inhibition of L-AlaDH, of the uncompetitive type,
by 3-fluoropyruvate is explained by the formation of an
NADH/3-fluoropyruvate complex. This complex binds NH4+,
the first product of the reaction and the complex thus
resulting (due to the equilibrium), then is changed (via
NADH-Schiff base complex) back to the intermediate
complex L-AlaDH-NADH-L-3-fluoroalanine (Figure 6).
Figure 6. Mechanistic proposal of the inhibition of the enzyme
by 3-fluoropyruvate.
pyruvate occurs, the possibility exits than these two products
inhibit the enzyme AlaDH with different strengths, 3-fluo-
Conclusions
+
ropyruvate being a stronger inhibitor than NH4 . To verify
The difference in the efficiency in two recycle systems,
using L-LDH and L-GluDH, was identified as being the result
of different potencies of the products that accumulate in both
systems, acting as inhibitors of L-AlaDH. It was demonstrated
that 3-fluoropyruvate is a much stronger inhibitor of L-AlaDH
this possibility inhibition studies of L-AlaDH by 3-fluoro-
+
pyruvate and NH4 were undertaken.
+
Figure 4 shows the results obtained when NH4 and
3-fluoropyruvate were tested as inhibitors, separately, of the
enzyme L-AlaDH. As shown in Figure 4, clearly 3-fluoro-
+
+
than NH4 . This fact, and not thermodynamic considerations,
pyruvate is a much stronger inhibitor of L-AlaDH than NH4 ;
explains the results obtained with both systems. The cascade
system developed which afforded the simultaneous produc-
tion of two fluorinated chiral products is strongly favored.
for instance with a concentration of 1.5 mM of the product
inhibitor, 3-fluoropyruvate caused almost a total inhibition
of the enzyme, whereas the same concentration of NH4
+
produced less than 20% of inhibition of L-AlaDH. The actual
values of residual activity of the enzyme at each concentra-
Received for review February 1, 2006.
OP060027O
(20) Baker, P. J.; Sawa, Y.; Shibata, H.; Sedelnikova, S. E.; Rice, D. W. Nat.
Struct. Biol. 1998, 5, 561.
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