- Email: [email protected]
Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase
Applied Radiation and Isotopes 123 (2017) 21–25
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase
MARK
⁎
Jolanta Szymańska-Majchrzaka, , Katarzyna Pałkab, Marianna Kańskaa a b
Department of Biochemistry, 2nd Faculty of Medicine, Medical University of Warsaw, 101 Żwirki i Wigury Ave., 02-089 Warsaw, Poland Department of Chemistry, University of Warsaw, 1 Pasteur Str., 02-093 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: L-alanine dehydrogenase Deuterium Isotope effect Fluorine UV–VIS spectrophotometry L-alanine
Synthesis of 3-fluoro-[2-2H]-L-alanine (3-F-[2H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H2O - KIE’s on Vmax: 1.1; on Vmax/KM: 1.2; for 3-F-L-Ala in 2 H2O – on Vmax: 1.4; on Vmax/KM: 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE’s on Vmax: 1.0; on Vmax/KM: 0.87; for 3-F-[2-2H]-L-Ala – on Vmax: 1.4; on Vmax/KM: 1.5). Studies explain some details of reaction mechanism.
1. Introduction Dehydrogenases are large group of enzymes belonging to the class of oxidoreductases which catalyse biochemical reactions of great significance. Enzyme L-alanine dehydrogenase (EC 1.4.1.1, AlaDH), a NAD+- dependent amino acid dehydrogenase, was found in various bacteria species i.a. Bacillus (Kim et al., 2000), Archaeoglobus, Streptomyces, and their catalytic along with kinetic properties and the structure have been well identified (Ohshima and Soda, 1990). AlaDH was also found and described in pathogenic Mycobacterium tuberculosis, what makes the enzyme a potential target for pathogen control by antibacterial compounds (Hutter and Singh, 1999). AlaDH, characterized by narrow substrate specificity, catalyses the reversible transformation of L-alanine into pyruvic acid. According to the direction of reaction, it acts towards pyruvate, 2-oxobutyrate, 2-oxovalerate and 3hydroxypyruvate in reductive amination, though in terms of the opposite reaction - oxidative deamination - the enzyme predominantly decomposes L-alanine (Ohshima and Soda, 1979) and some of its halogen derivatives (Gonçalves et al., 2003). In case of the stereochemistry of hydrogen transfer, AlaDH isolated from Bacillus subtilis shows pro-R stereospecificity, which indicates that hydrogen is transferred from the pro-R position at C-4 of the NADH nicotinamide ring to C-2 of pyruvate to form L-alanine (Alizade et al., 1975). Halogenated amino acids, including fluorinated, don’t occur naturally in living organisms, but they can be introduced like natural amino acids due to the similar van der Waals radius of fluorine (1.35 Å) and hydrogen (1.2 Å). High electronegativity of fluorine causes abnormalities in metabolism and functions of proteins containing unnatural ⁎
fluorinated amino acids. A few years ago it was discovered that compounds such as 3-fluoro-L-alanine (3-F-L-Ala) and 3-fluoro-D-alanine (3-F-D-Ala) exhibit antibacterial and antivirus properties (Kollonitsch and Barash, 1976) and they are broad spectrum antibiotics due to irreversible inactivation of alanine racemase - enzyme involved in biosynthesis of the bacterial cell wall (Ohshima et al., 1989; Esaki and Walsh, 1986). 3-F-L-Ala - an unnatural amino acid containing fluorine substituent, is the product of enzymatic conversion of 3-fluoropyruvic acid (3-F-PA) catalysed by AlaDH (Fig. 1). Enzymatic methods of syntheses are characterized with rapid, facile and specific course of action; therefore they are subjects of great interest in development of syntheses involving short-lived isotopes. Unnatural amino acids labelled with β+-emitting fluorine-18 can be applied as radioactive traces in Positron Emission Tomography (PET) for the diagnosis of cancer and neurodegenerative diseases (Laverman et al., 2002). 18F-Fluorinated amino acids such as 3-18F-L-Ala can be additional tools in cancer imaging by tracing of the protein biosynthesis, mainly by measuring amino acids transport rate to cancer cells via different mechanisms, providing fundamental components for tumour growth (Wang et al., 2012). In addition, fluorinated α-hydroxyacids and α-amino acids can be potentially highly versatile chiral building blocks for the asymmetric synthesis of compounds of pharmacological interest and they can be potential precursors of fluoroamine compounds (Gonçalves et al., 2000). Amino acids containing fluorine can be used in bioorganic applications such as biological traces, mechanistic probes (Qui et al., 2004) and as potential irreversible inhibitors of PLP-dependent en-
Corresponding author. E-mail address: [email protected] (J. Szymańska-Majchrzak).
http://dx.doi.org/10.1016/j.apradiso.2017.02.003 Received 9 September 2016; Received in revised form 12 January 2017; Accepted 2 February 2017 Available online 12 February 2017 0969-8043/ © 2017 Elsevier Ltd. All rights reserved.
Applied Radiation and Isotopes 123 (2017) 21–25
J. Szymańska-Majchrzak et al.
H F
NH2 OH
+
NAD+
+ H2O
L-alanine dehydrogenase
O OH +
F
O
NADH + H+ + NH4+
O
3-F-L-Ala
3-F-PA
Fig. 1. Reversible reaction of enzymatic biotransformation of 3-F-L-Ala catalysed by AlaDH from Bacillus subtilis.
an Amberlite IR 120H+ column (100×10 mm). Afterwards the column was washed with 500 mL of water in order to remove the salts and deuterium from labile positions of the product. 3-F-[2-2H]-L-Ala was eluted with 1 M NH3(aq), and collected as 7 mL fractions. The presence of the product in fractions was confirmed by TLC as mentioned before. The proper fractions were combined, concentrated under reduced pressure at 50 °C and lyophilized. As a result 5.7 mg (0.053 mmol) of 3-F-[2-2H]-L-Ala was obtained with 74% chem. yield. The extent of deuterium enrichment into α position of the product was determined by the 1H NMR spectrum (TMS, D2O, 500 MHz): δ [ppm] 4.1 (1H, H-α, 72% 2H), δ 4.9 (2H, H-β). The deficit of signal of proton at the α-carbon atom indicates 72% enrichment of deuterium at this position.
zymes like decarboxylases, transaminases and racemases (Seo et al., 2011). Despite the literature reports concerning the reactions catalysed by AlaDH, their mechanisms are still under investigation. In previous article (Szymańska and Kańska, 2013) we reported deuterium isotope effects for reductive amination of 3-F-PA catalysed by AlaDH. In this report we have studied some intrinsic details of enzymatic reaction catalysed by AlaDH involved in oxidative deamination of 3-F-L-Ala (AlaDH) (Fig. 1) using solvent (SIE) or kinetic (KIE) isotope effect methods. The numerical values of deuterium effects were determined using a non-competitive spectrophotometric method. This technique involves determining the ratios of rate constants using protium and deuterium. By labelling of the molecule at different specific positions and determining primary and secondary isotope effects it is possible to designate the rate determining step and characterize many details of the mechanism crucial for kinetics such as bond breaking/forming and the structure of active complex (Cook, 1991).
2.4. Kinetic assay 2.4.1. Determination of deuterium SIE’s and KIE’s for oxidative deamination of 3-F-L-Ala in reaction catalysed by AlaDH The kinetic experiments for studying SIE’s and KIE’s of oxidative deamination of 3-F-L-Ala were investigated using the following buffered solutions:
2. Experimental 2.1. Materials
a) b) c) d)
The enzyme L-alanine dehydrogenase (EC 1.4.1.1) from Bacillus subtilis, cofactors NAD+, NADH and other chemicals required for enzymatic synthesis and trail experiments, such as sodium 3-fluoropyruvate were from Sigma. Deuteriated water (99,9% 2H) and 30% KO2H/2H2O were from POLATOM (Poland). Thin layer chromatography (TLC) plates with UV indicator (DC-Plastikfolien Keiselgel 60 F254) and Amberlite IR-120 (Na+) were from Merck and Aldrich respectively. Selectively deuteriated cofactor [(4R)-2H]-NADH (100% 2H incorporation) was obtained using enzymatic method described in the recent article (Szymańska and Kańska, 2014).
50 mM carbonate buffer, pH 10.2 adjusted with 25 mM NaHCO3, 6 mM solution of 3-F-L-Ala or 3-F-[2-2H]-L-Ala, 15 mM solution of NAD+, −1 L-alanine dehydrogenase solution (1.1 U mL ).
Each kinetic experiment consisted of six runs for varying concentrations of 3-F-L-Ala or 3-F-[2-2H]-L-Ala. All measurements were performed using UV–Vis spectroscopy in 3 mL quartz cuvettes, filled with appropriate volumes of the buffered solutions of reagents. The final concentrations of the substrate ranged from 0.4 to 0.9 mM with 0.1 mM intervals. Afterwards, the exact volumes of NAD+ and AlaDH solutions were added to acquire 1.0 mM and 0.2 U mL−1 concentrations, respectively. Finally, each cuvette was filled with carbonate buffer up to 3 mL. The progress of oxidative deamination was registered according to the increasing absorbance measured spectrophotometrically at λmax=340 nm for 40 min (1 min intervals) as a result of forming NADH cofactor.
2.2. Methods The proton nuclear magnetic resonance (1H NMR) spectra were recorded in 2H2O using tetramethylsilane (TMS) as an internal standard on Varian 500 MHz Unity-plus spectrometer. The extent of deuterium incorporation was determined from 1H NMR spectrum. The kinetic assays were performed using Shimadzu-UV-1800 spectrophotometer. The reaction progress was checked by TLC (thin layer chromatography) using silica gel plates and ethanol/ammonia, 4:1; v/v as a developing solvent. 0,1% ninhydrin in ethanol and UV lamp were the methods of visualization of chromatograms.
2.5. SIE and KIE assays The enzymatic oxidative deamination of 3-F-L-Ala (Fig. 1) and its deuterium derivative (Fig. 2) was carried out at room temperature, and its kinetics was studied spectrophotometrically as described in Section 2.4. The kinetic parameters of Michaelis-Menten Eq. (3.1): Vmax and KM in all experiments were obtained using an indirect spectrophotometric method by measuring the increasing absorbance of reduced form of cofactor NADH showing maximum at λmax=340 nm, according to oxidative deamination of 3-F-L-Ala during the progress of reaction. SIE’s and KIE’s for enzymatic reaction catalysed by AlaDH (Fig. 1) were determined by a non-competitive method (Parkin, 1991). Data analysis was carried out using graphical representation of Lineweaver-Burk equation of enzyme kinetics. Parameters Vmax and KM were calculated using Lineweaver-Burk double reciprocal plot from the experimentally obtained reaction rates at varied concentrations of each substrate. The values of isotope effects were determined from initial rates (υ)
2.3. Synthesis of 3-F-[2-2H]-L-Ala To the 10 mL vial containing 9.2 mg (0.072 mmol) of sodium 3fluoropyruvate and 52 mg (0.074 mmol) of [(4R)-2H]-NADH (enriched in near 100% in the pro-R-position), 7 U of enzyme AlaDH was added and solubilized in 4 mL of 0.3 M deuteriated ammonium buffer (NH4Cl/ NH3·H2O) adjusted to pD 9.2 with 30% KO2H/2H2O. The reaction mixture was incubated at 30 °C for 24 h and its progress was monitored by TLC. The reaction was quenched by acidification with concentrated hydrochloric acid to pH~2 and the denaturized enzyme was removed by centrifugation. Then, the remaining 3-F-PA was extracted with 8×1 mL portions of diethyl ether and the mixture was loaded onto 22
Applied Radiation and Isotopes 123 (2017) 21–25
J. Szymańska-Majchrzak et al.
Fig. 2. Synthesis of 3-F-[2-2H]-L-Ala by reductive amination of 3-F-PA catalysed by AlaDH (R – the rest of cofactor NAD+/NADH).
according to the higher KD2O constant (Gary et al., 1964) and the source of deuterium was selectively labelled cofactor [(4R)-2H]-NADH (Szymańska and Kańska, 2014). On account of pro-R stereospecificity of AlaDH the deuterium from the 4R position of nicotinamide ring of cofactor was exclusively transferred to 3-F-PA forming 3-F-[2-2H]-L-Ala. The progress of reductive amination was monitored by TLC. The obtained product was separated and purified by extraction and ion exchange column chromatography. The chemical structure and the degree of deuterium enrichment – 72% – was confirmed by 1H NMR spectrum by measuring an integration of the signal for proton at α-C position in 3-F-[2-2H]-L-Ala.
for above mentioned isotopologue and calculated from Eq. (3.2) (Papajak et al., 2006):
⎡V ⎤ KM =[S ] ⎢ max −1⎥ , ⎣ υ ⎦ υ=
(3.1)
[S ] ⎛ 1 ⎜V K ⎝ max M
+
[S ] ⎞ ⎟ Vmax ⎠
(3.2)
where KM is the Michaelis constant, [S] – concentration of the substrate (S > > concentration of enzyme), Vmax – maximum velocity and υ – initial rate.
3.2. Determination of isotope effects of deuterium
2.5.1. SIE assay For SIE measurement of the reaction of oxidative deamination catalysed by AlaDH, the carbonate buffer 50 mM pH 10.2 (solution a) in Section 2.4.1) was prepared both in normal and fully deuteriated buffer media. For the latter, carbonate buffer was prepared using 2H2O, and adjusted to pD 10.6 using NaHCO3/2H2O. All reagents and enzymes listed in solutions b) – d) were dissolved in the respective buffers, and kinetics was measured to determine the rate of the reaction in each buffer. The SIE values were calculated by dividing the kinetics parameters Vmax and Vmax / KM for the reaction carried out with 3-F-L-Ala obtained in D and (Vmax / KM ) D the carbonate buffer by the kinetics parameters Vmax obtained in the fully deuteriated carbonate buffer, (Eqs. 3.3 and 3.4):
SIE =
Vmax D Vmax
Calculated values of isotope effects are shown in Table 1. Experimental errors were calculated with Student’s t-distribution for 95% confidence interval. In most cases normal isotope effects of deuterium were observed. Obtained values indicate that the reaction rate involving heavier isotope are lower than with lighter isotope. That suggests that breaking of C-2H bond is more difficult and requires more energy than in case of C-1H bond. As for determining of KIE’s for oxidative deamination in protonated and deuteriated buffer, 3-F-[2-2H]-L-alanine (72% 2H enrichment) was used, then the experimental data of SIE’s and KIE’s were approximated to the values corresponding to 100% of deuterium incorporation. For this correction (Parkin, 1991) Eq. (3.5) was used:
α = p (αp − 1) + 1
(3.3)
(3.5)
where: p – degree of enrichment with H; α – corrected isotope effect to 100% of deuterium incorporation (p=1); αp – measured isotope effect for the species with p degree of enrichment with 2H;. The experimental and corrected values of the isotope effects are given in Table 1 and in this certain case, the differences between the experimental and corrected values of obtained effects were rather small and mostly within the range of experimental errors. Determined values of KIE’s on Vmax indicate that the transfer of hydride from substrate to coenzyme NAD+, involving the formation of protonated iminopyruvate (Fig. 3), is an isotope-sensitive step. The values of KIE on Vmax/KM, have grater values than KIE on Vmax, what suggests that the transfer of hydride is a partially-limiting step and the reaction rate is limited by the release of product – NADH (Grimshaw and Cleland, Grimshaw and Cleland, 1981a, Grimshaw and Cleland, 1981b.). Obtained results of isotope effects for 3-F-L-Ala and other mechanistic studies of L-Ala described in recent article (Szymańska and Kańska, 2014) suggests that the rate-determining step in oxidative deamination catalysed by AlaDH is isomerization of AlaDH-NAD+ complex to a form that is capable of binding the reactant (Grimshaw and Cleland, 1981a, 1981b; Weiss et al., 1988). Calculated values of isotope effects in oxidative deamination of 3-F-L-Ala show that fluorine constituent doesn’t cause the significant change in the area of bonds that are being converted. Values of effects less than one are characteristic for 2° KIE and they can be a result of changing of hybridization from sp3 into sp2 during the forming of active complex in the rate determining step. Deuteriated solvent which compose the reaction medium in oxidative deamination catalysed by AlaDH only slightly affects the conversion of [E-S] complex into [E-P] complex and 2
SIE =
Vmax / KM (Vmax / KM ) D
(3.4)
This method of determining SIE’s was applied for 3-F-L-Ala and its deuteriated derivative 3-F-[2-2H]-L-Ala. 2.5.2. KIE assay The reaction rate was determined for oxidation catalysed by AlaDH of 3-F-L-Ala and stereospecifically deuterium-labelled 3-F-[2-2H]-L-Ala in carbonate buffer 50 mM pH 10.2 in normal water and in fully deuteriated (pD 10.6). The KIE values were calculated by dividing the kinetics parameters Vmax and Vmax /KM for the reaction carried out with 3-F-L-Ala and deuteriated 3-F-[2-2H]-L-Ala, obtained according to procedure described in Section 2.3, by the kinetics parameters VD max and (Vmax / KM ) D obtained in the normal and fully deuteriated carbonate buffer, (Eqs. 3.3 and 3.4): 3. Results and discussion 3.1. Synthesis The unnatural fluorinated amino acid – 3-fluoro-L-alanine was synthesized as was described in the recent article (Szymańska and Kańska, 2013). In this paper we have obtained 3-F-L-Ala labelled with deuterium in the α-C position as a result of enzymatic reductive amination of 3-F-PA. The reaction was catalysed by L-alanine dehydrogenase (EC 1.4.1.1) from Bacillus subtilis as presented at Fig. 3. The synthesis was carried out at 30ºC in ammonium buffer (pD 9.2 – 23
Applied Radiation and Isotopes 123 (2017) 21–25
J. Szymańska-Majchrzak et al.
Fig. 3. Proposed mechanism of oxidative deamination of L-Ala catalysed by AlaDH: (1) forming of protonated iminopyruvate involving the NAD+ reduction, (2) forming of carbinolamine as a result of nucleophilic attack by a molecule of water, (3) conversion leading to ammonia detachment, (4) releasing of pyruvate, ammonia and NADH (Brunhuber and Blanchard, 1994).
1) transfer of hydride is a partially-limiting step and the reaction rate is limited by the release of product – NADH; 2) fluorine constituent doesn’t cause the significant change in the area of bonds that are being converted; 3) deuteriated solvent present in the reaction medium only slightly affects the conversion of [E-S] complex into [E-P] complex.
Table 1 KIE and SIE values for oxidative deamination of 3-F-L-Ala and 3-F-[2-2H]-L-Ala. Solvent isotope effects on Vmax
on Vmax
on Vmax/KM
(corr)
3-F-L-Ala 3-F-[2-2H]-LAla
1.0 ± 0.2 1.5 ± 0.2
– 1.4 ± 0.2
on Vmax/KM (corr)
0.87 ± 0.06 1.7 ± 0.2
– 1.5 ± 0.2
Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Kinetic isotope effects on Vmax
on Vmax
on Vmax/KM
(corr)
3-F-L-Ala in H2O 3-F-L-Ala in 2 H2O
on Vmax/KM
References
(corr)
1.2 ± 0.1
1.1 ± 0.1
1.3 ± 0.3
1.2 ± 0.3
1.6 ± 0.1
1.4 ± 0.1
2.5 ± 0.3
2.1 ± 0.3
Alizade, M.A., Bressler, R., Brendel, K., 1975. Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)-alanine dehydrogenase from Bacillus subtilis. Biochim. Biophys. Acta 397, 5–8. Brunhuber, N.M.W., Blanchard, J.S., 1994. The biochemistry and enzymology of amino acid dehydrogenases. Crit. Rev. Biochem. Mol. Biol. 29 (6), 415–467. Cook, P.F., 1991. Kinetic and regulatory mechanism of enzymes from isotope effects. In: Cook, P.F. (Ed.), Enzyme Mechanism From Isotope Effects. CRS, Boca Raton, Boston, London, pp. 203–230. Esaki, N., Walsh, C.T., 1986. Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene. Biochemistry 25 (11), 3261–3267. Gary, R., Bates, R.G., Robinson, R.A., 1964. Second dissociation constant of deuteriophosphoric acid in deuterium oxide from 5 to 50 °C: standardization of pD scale. J. Phys. Chem. 68, 3806. Gonçalves, L.P.B., Antunes, O.A.C., Pinto, G.F., Oestreicher, E.G., 2000. Simultaneous enzymatic synthesis of (S)-3-fluoroalanine and (R)-3-fluorolactic acid. Tetrahedron.: Asymmetry 11, 1465–1468. Gonçalves, L.P.B., Antunes, O.A.C., Pinto, G.F., Oestreicher, E.G., 2003. Kinetic aspects involved in the simultaneous enzymatic synthesis of (S)-3-fluoroalanine and (R)-3fluorolactic acid. J. Fluor. Chem. 124, 219–227. Grimshaw, C.E., Cleland, W.W., 1981a. Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase. Biochemistry 20, 5655–5661.
Note: Means and SD’s were calculated from six kinetic measurements
dissociation of product from enzyme is slower than conversion of reactant into product (Schowen, 1972). 4. Conclusions Reductive amination catalysed by AlaDH involving specifically deuteriated cofactor [(4R)-2H]-NADH allows to synthesize deuteriated derivative of 3-F-L-Ala - 3-F-[2-2H]-L-Ala. The same enzyme – AlaDH – was used to mechanistic studies of oxidative deamination of 3-F-L-Ala. The numerical values of SIE and KIE were determined using a non-competitive spectrophotometric method. Obtained values indicate that: 24
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Parkin, D.W., 1991. Methods for determination of competitive and noncompetitive kinetic isotope effects. In: Cook, P.F. (Ed.), Enzyme Mechanism from Isotope Effects. CRS, Boca Raton, Boston, London, pp. 269–290. Qui, X.-L., Meng, W.-D., Quing, F.-L., 2004. Synthesis of fluorinated amino acids. Tetrahedron 60, 6711–6745. Schowen, R.L., 1972. Mechanistic deductions from solvent isotope effect. Prog. Phys. Org. Chem. 9, 275–332. Seo, Y.-M., Khang, Y.-H., Yun, H., 2011. Kinetic resolution of 3-fluoroalanine using a fusion protein of D-amino acid oxidase with Vitroscilla hemoglobin. Biosci. Biotechnol. Biochem 75 (4), 820–822. Szymańska, J., Kańska, M., 2013. Deuterium isotope effects in reduction of βfluoropyruvic acid catalyzed by L-alanine dehydrogenase. Copernic. Lett. 4, 103–109. http://dx.doi.org/10.12775/CL.2013.012. Szymańska, J., Kańska, M., 2014. Deuterium isotope effects in oxidative deamination of LAlanine catalyzed by L-Alanine dehydrogenase. J. Chem. Chem. Eng. 8, 145–150. Wang, L., Zha, Z., Qu, W., Qiao, H., Lieberman, B.P., Plössl, K., Kung, H.F., 2012. Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents. Nucl. Med. Biol. 39 (7), 933–943. Weiss, P.M., Chen, C.-Y., Cleland, W.W., Cook, P.F., 1988. Use of primary deuterium and 15 N isotope effects to deduce the relative rates of steps in the mechanism of alanine and glutamate dehydrogenases. Biochemistry 27, 4814–4822.
Grimshaw, C.E., Cleland, W.W., 1981b. Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase. Biochemistry 20, 5650–5655. Hutter, B., Singh, M., 1999. Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase. Biochem. J. 343, 669–672. Kim, S., Kim, Y.J., Seo, M.R., Jhun, B.S., 2000. Regulatory mechanism of L-Alanine dehydrogenase from Bacillus subtilis. Bull. Korean Chem. Soc. 21, 1217–1221. Kollonitsch, J., Barash, L., 1976. Organofluorine synthesis via photofluorination: 3-fluorod-alanine and 2-deuterio analogue, antibacterials related to the bacterial cell wall. J. Am. Chem. Soc. 98 (18), 5591–5593. Laverman, P., Boerman, O.C., Corstens, F.H.M., Oyen, W.J.G., 2002. Fluorinated amino acids for tumour imaging with positron emission tomography. Eur. J. Nucl. Med. 29, 681–690. Ohshima, T., Soda, K., 1979. Purification and properties of alanine dehydrogenase from Bacillus sphaericus. Eur. J. Biochem 100, 29–39. Ohshima, T., Soda, K., 1990. Biochemistry and biotechnology of amino acid dehydrogenases. Adv. Biochem. Eng. Biotechnol. 42, 187–209. Ohshima, T., Wandrey, C., Conrad, D., 1989. Continuous production of 3-fluoro-L-alanine with alanine dehydrogenase. Biotechnol. Bioeng. 34, 394–397. Papajak, E., Kwiecień, R.A., Rudziński, J., Sicińska, D., Kamiński, R., Szadkowski, Ł., Kurihara, T., Esaki, N., Paneth, P., 2006. Mechanism of the reaction catalyzed by DL2-Haloacid dehalogenase as determined from kinetic isotope effects. Biochemistry 45, 6012–6017.
25
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase
MARK
⁎
Jolanta Szymańska-Majchrzaka, , Katarzyna Pałkab, Marianna Kańskaa a b
Department of Biochemistry, 2nd Faculty of Medicine, Medical University of Warsaw, 101 Żwirki i Wigury Ave., 02-089 Warsaw, Poland Department of Chemistry, University of Warsaw, 1 Pasteur Str., 02-093 Warsaw, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: L-alanine dehydrogenase Deuterium Isotope effect Fluorine UV–VIS spectrophotometry L-alanine
Synthesis of 3-fluoro-[2-2H]-L-alanine (3-F-[2H]-L-Ala) in reductive amination of 3-fluoropyruvic acid catalysed by L-alanine dehydrogenase (AlaDH) was described. Fluorine derivative was used to study oxidative deamination catalysed by AlaDH applied kinetic (for 3-F-L-Ala in H2O - KIE’s on Vmax: 1.1; on Vmax/KM: 1.2; for 3-F-L-Ala in 2 H2O – on Vmax: 1.4; on Vmax/KM: 2.1) and solvent isotope effect methods (for 3-F-L-Ala - SIE’s on Vmax: 1.0; on Vmax/KM: 0.87; for 3-F-[2-2H]-L-Ala – on Vmax: 1.4; on Vmax/KM: 1.5). Studies explain some details of reaction mechanism.
1. Introduction Dehydrogenases are large group of enzymes belonging to the class of oxidoreductases which catalyse biochemical reactions of great significance. Enzyme L-alanine dehydrogenase (EC 1.4.1.1, AlaDH), a NAD+- dependent amino acid dehydrogenase, was found in various bacteria species i.a. Bacillus (Kim et al., 2000), Archaeoglobus, Streptomyces, and their catalytic along with kinetic properties and the structure have been well identified (Ohshima and Soda, 1990). AlaDH was also found and described in pathogenic Mycobacterium tuberculosis, what makes the enzyme a potential target for pathogen control by antibacterial compounds (Hutter and Singh, 1999). AlaDH, characterized by narrow substrate specificity, catalyses the reversible transformation of L-alanine into pyruvic acid. According to the direction of reaction, it acts towards pyruvate, 2-oxobutyrate, 2-oxovalerate and 3hydroxypyruvate in reductive amination, though in terms of the opposite reaction - oxidative deamination - the enzyme predominantly decomposes L-alanine (Ohshima and Soda, 1979) and some of its halogen derivatives (Gonçalves et al., 2003). In case of the stereochemistry of hydrogen transfer, AlaDH isolated from Bacillus subtilis shows pro-R stereospecificity, which indicates that hydrogen is transferred from the pro-R position at C-4 of the NADH nicotinamide ring to C-2 of pyruvate to form L-alanine (Alizade et al., 1975). Halogenated amino acids, including fluorinated, don’t occur naturally in living organisms, but they can be introduced like natural amino acids due to the similar van der Waals radius of fluorine (1.35 Å) and hydrogen (1.2 Å). High electronegativity of fluorine causes abnormalities in metabolism and functions of proteins containing unnatural ⁎
fluorinated amino acids. A few years ago it was discovered that compounds such as 3-fluoro-L-alanine (3-F-L-Ala) and 3-fluoro-D-alanine (3-F-D-Ala) exhibit antibacterial and antivirus properties (Kollonitsch and Barash, 1976) and they are broad spectrum antibiotics due to irreversible inactivation of alanine racemase - enzyme involved in biosynthesis of the bacterial cell wall (Ohshima et al., 1989; Esaki and Walsh, 1986). 3-F-L-Ala - an unnatural amino acid containing fluorine substituent, is the product of enzymatic conversion of 3-fluoropyruvic acid (3-F-PA) catalysed by AlaDH (Fig. 1). Enzymatic methods of syntheses are characterized with rapid, facile and specific course of action; therefore they are subjects of great interest in development of syntheses involving short-lived isotopes. Unnatural amino acids labelled with β+-emitting fluorine-18 can be applied as radioactive traces in Positron Emission Tomography (PET) for the diagnosis of cancer and neurodegenerative diseases (Laverman et al., 2002). 18F-Fluorinated amino acids such as 3-18F-L-Ala can be additional tools in cancer imaging by tracing of the protein biosynthesis, mainly by measuring amino acids transport rate to cancer cells via different mechanisms, providing fundamental components for tumour growth (Wang et al., 2012). In addition, fluorinated α-hydroxyacids and α-amino acids can be potentially highly versatile chiral building blocks for the asymmetric synthesis of compounds of pharmacological interest and they can be potential precursors of fluoroamine compounds (Gonçalves et al., 2000). Amino acids containing fluorine can be used in bioorganic applications such as biological traces, mechanistic probes (Qui et al., 2004) and as potential irreversible inhibitors of PLP-dependent en-
Corresponding author. E-mail address: [email protected] (J. Szymańska-Majchrzak).
http://dx.doi.org/10.1016/j.apradiso.2017.02.003 Received 9 September 2016; Received in revised form 12 January 2017; Accepted 2 February 2017 Available online 12 February 2017 0969-8043/ © 2017 Elsevier Ltd. All rights reserved.
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H F
NH2 OH
+
NAD+
+ H2O
L-alanine dehydrogenase
O OH +
F
O
NADH + H+ + NH4+
O
3-F-L-Ala
3-F-PA
Fig. 1. Reversible reaction of enzymatic biotransformation of 3-F-L-Ala catalysed by AlaDH from Bacillus subtilis.
an Amberlite IR 120H+ column (100×10 mm). Afterwards the column was washed with 500 mL of water in order to remove the salts and deuterium from labile positions of the product. 3-F-[2-2H]-L-Ala was eluted with 1 M NH3(aq), and collected as 7 mL fractions. The presence of the product in fractions was confirmed by TLC as mentioned before. The proper fractions were combined, concentrated under reduced pressure at 50 °C and lyophilized. As a result 5.7 mg (0.053 mmol) of 3-F-[2-2H]-L-Ala was obtained with 74% chem. yield. The extent of deuterium enrichment into α position of the product was determined by the 1H NMR spectrum (TMS, D2O, 500 MHz): δ [ppm] 4.1 (1H, H-α, 72% 2H), δ 4.9 (2H, H-β). The deficit of signal of proton at the α-carbon atom indicates 72% enrichment of deuterium at this position.
zymes like decarboxylases, transaminases and racemases (Seo et al., 2011). Despite the literature reports concerning the reactions catalysed by AlaDH, their mechanisms are still under investigation. In previous article (Szymańska and Kańska, 2013) we reported deuterium isotope effects for reductive amination of 3-F-PA catalysed by AlaDH. In this report we have studied some intrinsic details of enzymatic reaction catalysed by AlaDH involved in oxidative deamination of 3-F-L-Ala (AlaDH) (Fig. 1) using solvent (SIE) or kinetic (KIE) isotope effect methods. The numerical values of deuterium effects were determined using a non-competitive spectrophotometric method. This technique involves determining the ratios of rate constants using protium and deuterium. By labelling of the molecule at different specific positions and determining primary and secondary isotope effects it is possible to designate the rate determining step and characterize many details of the mechanism crucial for kinetics such as bond breaking/forming and the structure of active complex (Cook, 1991).
2.4. Kinetic assay 2.4.1. Determination of deuterium SIE’s and KIE’s for oxidative deamination of 3-F-L-Ala in reaction catalysed by AlaDH The kinetic experiments for studying SIE’s and KIE’s of oxidative deamination of 3-F-L-Ala were investigated using the following buffered solutions:
2. Experimental 2.1. Materials
a) b) c) d)
The enzyme L-alanine dehydrogenase (EC 1.4.1.1) from Bacillus subtilis, cofactors NAD+, NADH and other chemicals required for enzymatic synthesis and trail experiments, such as sodium 3-fluoropyruvate were from Sigma. Deuteriated water (99,9% 2H) and 30% KO2H/2H2O were from POLATOM (Poland). Thin layer chromatography (TLC) plates with UV indicator (DC-Plastikfolien Keiselgel 60 F254) and Amberlite IR-120 (Na+) were from Merck and Aldrich respectively. Selectively deuteriated cofactor [(4R)-2H]-NADH (100% 2H incorporation) was obtained using enzymatic method described in the recent article (Szymańska and Kańska, 2014).
50 mM carbonate buffer, pH 10.2 adjusted with 25 mM NaHCO3, 6 mM solution of 3-F-L-Ala or 3-F-[2-2H]-L-Ala, 15 mM solution of NAD+, −1 L-alanine dehydrogenase solution (1.1 U mL ).
Each kinetic experiment consisted of six runs for varying concentrations of 3-F-L-Ala or 3-F-[2-2H]-L-Ala. All measurements were performed using UV–Vis spectroscopy in 3 mL quartz cuvettes, filled with appropriate volumes of the buffered solutions of reagents. The final concentrations of the substrate ranged from 0.4 to 0.9 mM with 0.1 mM intervals. Afterwards, the exact volumes of NAD+ and AlaDH solutions were added to acquire 1.0 mM and 0.2 U mL−1 concentrations, respectively. Finally, each cuvette was filled with carbonate buffer up to 3 mL. The progress of oxidative deamination was registered according to the increasing absorbance measured spectrophotometrically at λmax=340 nm for 40 min (1 min intervals) as a result of forming NADH cofactor.
2.2. Methods The proton nuclear magnetic resonance (1H NMR) spectra were recorded in 2H2O using tetramethylsilane (TMS) as an internal standard on Varian 500 MHz Unity-plus spectrometer. The extent of deuterium incorporation was determined from 1H NMR spectrum. The kinetic assays were performed using Shimadzu-UV-1800 spectrophotometer. The reaction progress was checked by TLC (thin layer chromatography) using silica gel plates and ethanol/ammonia, 4:1; v/v as a developing solvent. 0,1% ninhydrin in ethanol and UV lamp were the methods of visualization of chromatograms.
2.5. SIE and KIE assays The enzymatic oxidative deamination of 3-F-L-Ala (Fig. 1) and its deuterium derivative (Fig. 2) was carried out at room temperature, and its kinetics was studied spectrophotometrically as described in Section 2.4. The kinetic parameters of Michaelis-Menten Eq. (3.1): Vmax and KM in all experiments were obtained using an indirect spectrophotometric method by measuring the increasing absorbance of reduced form of cofactor NADH showing maximum at λmax=340 nm, according to oxidative deamination of 3-F-L-Ala during the progress of reaction. SIE’s and KIE’s for enzymatic reaction catalysed by AlaDH (Fig. 1) were determined by a non-competitive method (Parkin, 1991). Data analysis was carried out using graphical representation of Lineweaver-Burk equation of enzyme kinetics. Parameters Vmax and KM were calculated using Lineweaver-Burk double reciprocal plot from the experimentally obtained reaction rates at varied concentrations of each substrate. The values of isotope effects were determined from initial rates (υ)
2.3. Synthesis of 3-F-[2-2H]-L-Ala To the 10 mL vial containing 9.2 mg (0.072 mmol) of sodium 3fluoropyruvate and 52 mg (0.074 mmol) of [(4R)-2H]-NADH (enriched in near 100% in the pro-R-position), 7 U of enzyme AlaDH was added and solubilized in 4 mL of 0.3 M deuteriated ammonium buffer (NH4Cl/ NH3·H2O) adjusted to pD 9.2 with 30% KO2H/2H2O. The reaction mixture was incubated at 30 °C for 24 h and its progress was monitored by TLC. The reaction was quenched by acidification with concentrated hydrochloric acid to pH~2 and the denaturized enzyme was removed by centrifugation. Then, the remaining 3-F-PA was extracted with 8×1 mL portions of diethyl ether and the mixture was loaded onto 22
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Fig. 2. Synthesis of 3-F-[2-2H]-L-Ala by reductive amination of 3-F-PA catalysed by AlaDH (R – the rest of cofactor NAD+/NADH).
according to the higher KD2O constant (Gary et al., 1964) and the source of deuterium was selectively labelled cofactor [(4R)-2H]-NADH (Szymańska and Kańska, 2014). On account of pro-R stereospecificity of AlaDH the deuterium from the 4R position of nicotinamide ring of cofactor was exclusively transferred to 3-F-PA forming 3-F-[2-2H]-L-Ala. The progress of reductive amination was monitored by TLC. The obtained product was separated and purified by extraction and ion exchange column chromatography. The chemical structure and the degree of deuterium enrichment – 72% – was confirmed by 1H NMR spectrum by measuring an integration of the signal for proton at α-C position in 3-F-[2-2H]-L-Ala.
for above mentioned isotopologue and calculated from Eq. (3.2) (Papajak et al., 2006):
⎡V ⎤ KM =[S ] ⎢ max −1⎥ , ⎣ υ ⎦ υ=
(3.1)
[S ] ⎛ 1 ⎜V K ⎝ max M
+
[S ] ⎞ ⎟ Vmax ⎠
(3.2)
where KM is the Michaelis constant, [S] – concentration of the substrate (S > > concentration of enzyme), Vmax – maximum velocity and υ – initial rate.
3.2. Determination of isotope effects of deuterium
2.5.1. SIE assay For SIE measurement of the reaction of oxidative deamination catalysed by AlaDH, the carbonate buffer 50 mM pH 10.2 (solution a) in Section 2.4.1) was prepared both in normal and fully deuteriated buffer media. For the latter, carbonate buffer was prepared using 2H2O, and adjusted to pD 10.6 using NaHCO3/2H2O. All reagents and enzymes listed in solutions b) – d) were dissolved in the respective buffers, and kinetics was measured to determine the rate of the reaction in each buffer. The SIE values were calculated by dividing the kinetics parameters Vmax and Vmax / KM for the reaction carried out with 3-F-L-Ala obtained in D and (Vmax / KM ) D the carbonate buffer by the kinetics parameters Vmax obtained in the fully deuteriated carbonate buffer, (Eqs. 3.3 and 3.4):
SIE =
Vmax D Vmax
Calculated values of isotope effects are shown in Table 1. Experimental errors were calculated with Student’s t-distribution for 95% confidence interval. In most cases normal isotope effects of deuterium were observed. Obtained values indicate that the reaction rate involving heavier isotope are lower than with lighter isotope. That suggests that breaking of C-2H bond is more difficult and requires more energy than in case of C-1H bond. As for determining of KIE’s for oxidative deamination in protonated and deuteriated buffer, 3-F-[2-2H]-L-alanine (72% 2H enrichment) was used, then the experimental data of SIE’s and KIE’s were approximated to the values corresponding to 100% of deuterium incorporation. For this correction (Parkin, 1991) Eq. (3.5) was used:
α = p (αp − 1) + 1
(3.3)
(3.5)
where: p – degree of enrichment with H; α – corrected isotope effect to 100% of deuterium incorporation (p=1); αp – measured isotope effect for the species with p degree of enrichment with 2H;. The experimental and corrected values of the isotope effects are given in Table 1 and in this certain case, the differences between the experimental and corrected values of obtained effects were rather small and mostly within the range of experimental errors. Determined values of KIE’s on Vmax indicate that the transfer of hydride from substrate to coenzyme NAD+, involving the formation of protonated iminopyruvate (Fig. 3), is an isotope-sensitive step. The values of KIE on Vmax/KM, have grater values than KIE on Vmax, what suggests that the transfer of hydride is a partially-limiting step and the reaction rate is limited by the release of product – NADH (Grimshaw and Cleland, Grimshaw and Cleland, 1981a, Grimshaw and Cleland, 1981b.). Obtained results of isotope effects for 3-F-L-Ala and other mechanistic studies of L-Ala described in recent article (Szymańska and Kańska, 2014) suggests that the rate-determining step in oxidative deamination catalysed by AlaDH is isomerization of AlaDH-NAD+ complex to a form that is capable of binding the reactant (Grimshaw and Cleland, 1981a, 1981b; Weiss et al., 1988). Calculated values of isotope effects in oxidative deamination of 3-F-L-Ala show that fluorine constituent doesn’t cause the significant change in the area of bonds that are being converted. Values of effects less than one are characteristic for 2° KIE and they can be a result of changing of hybridization from sp3 into sp2 during the forming of active complex in the rate determining step. Deuteriated solvent which compose the reaction medium in oxidative deamination catalysed by AlaDH only slightly affects the conversion of [E-S] complex into [E-P] complex and 2
SIE =
Vmax / KM (Vmax / KM ) D
(3.4)
This method of determining SIE’s was applied for 3-F-L-Ala and its deuteriated derivative 3-F-[2-2H]-L-Ala. 2.5.2. KIE assay The reaction rate was determined for oxidation catalysed by AlaDH of 3-F-L-Ala and stereospecifically deuterium-labelled 3-F-[2-2H]-L-Ala in carbonate buffer 50 mM pH 10.2 in normal water and in fully deuteriated (pD 10.6). The KIE values were calculated by dividing the kinetics parameters Vmax and Vmax /KM for the reaction carried out with 3-F-L-Ala and deuteriated 3-F-[2-2H]-L-Ala, obtained according to procedure described in Section 2.3, by the kinetics parameters VD max and (Vmax / KM ) D obtained in the normal and fully deuteriated carbonate buffer, (Eqs. 3.3 and 3.4): 3. Results and discussion 3.1. Synthesis The unnatural fluorinated amino acid – 3-fluoro-L-alanine was synthesized as was described in the recent article (Szymańska and Kańska, 2013). In this paper we have obtained 3-F-L-Ala labelled with deuterium in the α-C position as a result of enzymatic reductive amination of 3-F-PA. The reaction was catalysed by L-alanine dehydrogenase (EC 1.4.1.1) from Bacillus subtilis as presented at Fig. 3. The synthesis was carried out at 30ºC in ammonium buffer (pD 9.2 – 23
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Fig. 3. Proposed mechanism of oxidative deamination of L-Ala catalysed by AlaDH: (1) forming of protonated iminopyruvate involving the NAD+ reduction, (2) forming of carbinolamine as a result of nucleophilic attack by a molecule of water, (3) conversion leading to ammonia detachment, (4) releasing of pyruvate, ammonia and NADH (Brunhuber and Blanchard, 1994).
1) transfer of hydride is a partially-limiting step and the reaction rate is limited by the release of product – NADH; 2) fluorine constituent doesn’t cause the significant change in the area of bonds that are being converted; 3) deuteriated solvent present in the reaction medium only slightly affects the conversion of [E-S] complex into [E-P] complex.
Table 1 KIE and SIE values for oxidative deamination of 3-F-L-Ala and 3-F-[2-2H]-L-Ala. Solvent isotope effects on Vmax
on Vmax
on Vmax/KM
(corr)
3-F-L-Ala 3-F-[2-2H]-LAla
1.0 ± 0.2 1.5 ± 0.2
– 1.4 ± 0.2
on Vmax/KM (corr)
0.87 ± 0.06 1.7 ± 0.2
– 1.5 ± 0.2
Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Kinetic isotope effects on Vmax
on Vmax
on Vmax/KM
(corr)
3-F-L-Ala in H2O 3-F-L-Ala in 2 H2O
on Vmax/KM
References
(corr)
1.2 ± 0.1
1.1 ± 0.1
1.3 ± 0.3
1.2 ± 0.3
1.6 ± 0.1
1.4 ± 0.1
2.5 ± 0.3
2.1 ± 0.3
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Note: Means and SD’s were calculated from six kinetic measurements
dissociation of product from enzyme is slower than conversion of reactant into product (Schowen, 1972). 4. Conclusions Reductive amination catalysed by AlaDH involving specifically deuteriated cofactor [(4R)-2H]-NADH allows to synthesize deuteriated derivative of 3-F-L-Ala - 3-F-[2-2H]-L-Ala. The same enzyme – AlaDH – was used to mechanistic studies of oxidative deamination of 3-F-L-Ala. The numerical values of SIE and KIE were determined using a non-competitive spectrophotometric method. Obtained values indicate that: 24
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Grimshaw, C.E., Cleland, W.W., 1981b. Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase. Biochemistry 20, 5650–5655. Hutter, B., Singh, M., 1999. Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase. Biochem. J. 343, 669–672. Kim, S., Kim, Y.J., Seo, M.R., Jhun, B.S., 2000. Regulatory mechanism of L-Alanine dehydrogenase from Bacillus subtilis. Bull. Korean Chem. Soc. 21, 1217–1221. Kollonitsch, J., Barash, L., 1976. Organofluorine synthesis via photofluorination: 3-fluorod-alanine and 2-deuterio analogue, antibacterials related to the bacterial cell wall. J. Am. Chem. Soc. 98 (18), 5591–5593. Laverman, P., Boerman, O.C., Corstens, F.H.M., Oyen, W.J.G., 2002. Fluorinated amino acids for tumour imaging with positron emission tomography. Eur. J. Nucl. Med. 29, 681–690. Ohshima, T., Soda, K., 1979. Purification and properties of alanine dehydrogenase from Bacillus sphaericus. Eur. J. Biochem 100, 29–39. Ohshima, T., Soda, K., 1990. Biochemistry and biotechnology of amino acid dehydrogenases. Adv. Biochem. Eng. Biotechnol. 42, 187–209. Ohshima, T., Wandrey, C., Conrad, D., 1989. Continuous production of 3-fluoro-L-alanine with alanine dehydrogenase. Biotechnol. Bioeng. 34, 394–397. Papajak, E., Kwiecień, R.A., Rudziński, J., Sicińska, D., Kamiński, R., Szadkowski, Ł., Kurihara, T., Esaki, N., Paneth, P., 2006. Mechanism of the reaction catalyzed by DL2-Haloacid dehalogenase as determined from kinetic isotope effects. Biochemistry 45, 6012–6017.
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