Kinetic and solvent isotope effects on biotransformation of aromatic amino acids and their derivatives

Article abstract of DOI:10.1002/jlcr.3419

Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3′,4′-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3′,4′-dihydroxyphenylacelaldehyde (DOPAL) and 3′,4′-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5′-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2′-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.

Full text of DOI:10.1002/jlcr.3419

Special issue article
Received 04 March 2016,
Revised 09 May 2016,
Accepted 23 May 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3419
Kinetic and solvent isotope effects on
biotransformation of aromatic amino acids
and their derivatives
c
b
Marianna Kańska,a,b Jacek Jemielity, Małgorzata Pająk,
*
Katarzyna Pałka,b Katarzyna Podsadnia and Elżbieta Winnickab
Aromatic amino acids such as L-phenylalanine, L-tryptophan, 3′,4′-dihydroxy-L-phenylalanine (L-DOPA), and their derivatives
3′,4′-dihydroxyphenylacelaldehyde (DOPAL) and 3′,4′-dihydroxyphenylethanol (DOPET), play an essential role in human
metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and
in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these
metabolites draw biochemists’ and medical researchers’ attention. This study investigates the mechanisms of
biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The
overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation
of L-phenylalanine, 5′-chloro-L-tryptophan, and L-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine
ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the
deamination of 2′-chloro-L-phenylalanine in the presence of the enzyme L-phenylalanine dehydrogenase, as well as in the
conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been
determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard)
radiometric methods.
Keywords: DOPAL; enzymes; isotope effects; L-DOPA; L-phenylalanine; L-tryptophan
being the most representative and useful in the discussion of
Introduction
this investigation of KIE and SIE isotope effects determined for
The mechanistic studies of reactions catalyzed by some enzymes
the reaction catalyzed by PAL.
from the groups of lyases (phenylalanine ammonia lyase,
The tyrosine decarboxylase enzyme catalyzes the
tyrosine decarboxylase, and tryptophan indole-lyase), and
decarboxylation of 3′,4′-dihydroxy-L-phenylalanine (L-DOPA) to
dehydrogenases (L-phenyloalanine dehydrogenase, and alcohol
dopamine; see Scheme 2. Both these compounds, found in
dehydrogenase) using kinetic isotope effect (KIE) and solvent
nervous systems of mammals, are important neurotransmitters
and hormones.8 The disturbance in the production and
isotope effect (SIE) methods. The numerical values of KIE and
SIE are very helpful for clarifying the mechanistic intrinsic details
metabolism of dopamine and its derivatives leads to
development of many pathologies, including the neurological
disorders, such as Alzheimer’s, Parkinson’s diseases, and
schizophrenia. The incorrect metabolism of L-DOPA induces the
or for distinction between the alternative mechanisms.1
Phenylalanine ammonia lyase, PAL, (EC 4.3.1.5), an enzyme
commonly found in the higher plants and in some species of
fungi, plays an important role in the metabolism of plants. The
conversion of L-phenylalanine, L-Phe, into cinnamic acid is the
accumulation of some toxic metabolites, that is, 3′,4′-
dihydroxyphenylacetaldehyd
(DOPAL)
and
3′,4′-
first step in the biosynthesis of many important biological
phenylpropanoid derivatives such as lignins, flavonoids,
isoflavonoids, some of alkaloids, furanocoumarins, and the plant
hormones. It is also involved in the biosynthesis of flavonoid
pigments and of compounds protecting plants against harmful
pathogenic factors (e.g., UV) and defending against herbivores.2
PAL is used in experiments related to the treatment and
diagnosis of phenylketonuria3 and producing L-Phe from (E)-
cinnamic acid by addition of ammonia in vivo.4 Generally, PAL
dihydroxyphenylethanol (DOPET), which – combined with the
aDepartment of Biochemistry, Medical University of Warsaw, 2nd Faculty of
Medicine, 101 Zwirki i Wigury Av.,02-089 Warsaw, Poland
bDepartment of Chemistry, Warsaw University, 1 Pasteur Str.,02-093 Warsaw,
Poland
catalyzes the reversible elimination of ammonia from L-Phe cUniversity of Warsaw, Centre of New Technologies, 2c Banacha Str.,02-097
Warsaw, Poland
yelding (E)-cinnamic acid, Scheme 1.
The mechanism of ammonia elimination from L-Phe has
*Correspondence to: Marianna Kańska, Department of Chemistry, Warsaw
gained much attention and has been discussed by many
University, 1 Pasteur Str., 02-093 Warsaw, Poland.
research groups. This publication refers only to the ones,5–7 E-mail: mkanska@chem.uw.edu.pl.
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Copyright © 2016 John Wiley & Sons, Ltd.

M. Kańska et al.
metabolized by enforced side reactions12 to phenylpyruvate,
phenylacetate, and phenyl-L-lactate; see Scheme 4. The excess
of L-Phe in cells hinders the transport of other amino acids,
causing
a reduction in neurotransmitters and melanins
synthesis. PKU leads to mental retardation, brain damage, and
skin discoloration.
Scheme 1. The reversible reaction catalyzed by the enzyme phenylalanine
ammonia lyase (EC 4.3.1.5).
For this purpose, as mentioned earlier, the deuterium KIE
and SIE for 2′-chloro-[(3S)-2H]-L-Phe in the oxidative
deamination reaction catalyzed by PheDH were investigated;
see Scheme 5.
The results of studies of the KIE and SIE for the
aforementioned enzymatic reactions, useful for explaining some
details of their mechanisms, have not been published yet.
Experimental
Materials
The enzymes: PAL (EC 4.3.1.5) from Rhodotorula glutinis, Tyrosine
Decarboxylase (EC 4.1.1.25) from Streptococcus faecalis, Tyrosinase (EC
1.14.18.1) from Neurospora crassa, TPase (EC 4.1.99.1) from Escherichia.
coli, Lactate Dehydrogenase (EC 1.1.1.27) from rabbit muscle, PheDH
(EC 1.4.1.20) from Sporosarcina sp., ALDH (EC 1.1.1.2) from
Thermoanaerobium brockii, Malic Dehydrogenase (EC 1.1.1.40) from
chicken liver, Glucose Dehydrogenas (EC 1.1.1.47) from Pseudomonas
sp., as well as the cofactors: PLP (Pyridoxal 5′-Phosphate), NADH and
NAD+ (nicotinamide adenine dinucleotide – reduced and oxidized
forms), NADPH and NADP+ (nicotinamide adenine dinucleotide
phosphate – reduced and oxidized forms) were from Sigma.
Scheme 2. Incorrect metabolism of L-Tyr to DOPET.
imbalance of protective enzymes – may result in degeneration of
dopamine neurons in the brain.
Radioactive preparations, that is, [14C]KCN, [14C]BaCO3, and [1-14C]-
sodium acetate were supplied from Radioisotope Centre Polatom,
Świerk, Poland. [1-14C]- and [2-14C]-malonic acids and 3H2O were
purchased from NEN, Science Products Inc., Boston, USA. [1-14C]-L-
This research determines 14C KIE for the 1-position of L-DOPA
in its decarboxylation to dopamine, catalyzed by the tyrosine
decarboxylase enzyme (EC 4.1.1.25); see Scheme 2. The
deuterium KIE and SIE in reduction of DOPAL to DOPET in the
presence of a cofactor nicotinamide adenine dinucleotide
phosphate (NADPH), catalyzed by the alcohol dehydrogenase
3
tyrosine and H2O were from Moravec Biochemical, USA. The producers
of deuteriated substrates used in our experiments are listed as follows:
Heavy water (99.9% D) (Sigma, St. Louis, MO, USA); [1-2H]-D-glucose
(Aldrich); concentrated 2HCl/2H2O, 85% 2H3PO4/2H2O, and 30%
enzyme (ALDH, EC 1.1.1.2), have also been investigated; see KO2H/2H2O (POLATOM).
Liquid scintillations cocktails were produced by Rotiszint Eco Plus
Roth, Germany.
The other chemicals and solvents needed for trial synthesis, kinetics
runs, and separation and purification procedures were purchased from
Aldrich.
Scheme 2.
The mechanism of action of halogen derivatives of amino
acids and bioamines, labeled with the short-lived β+-isotopes
and used in medicinal diagnostics,9 is not fully understood
because the presence of halogen in their structure causes a
change in the catalytic activity of enzymes involved in their
degradation. Therefore, this research investigates the
mechanism of their biodegradation using the KIE and SIE
methods. The tryptophan indole-lyase enzyme (TPase, EC
4.1.99.1) catalyzes the chemical reversible reaction10; see
Scheme 3.
Methods
The radioactivity of all samples was determined using a liquid scintillation
counter (LISA, LSC PWW470, Germany) or Tri-Carb 2910 TR, PerkinElmer
(Waltham, MA, USA). In the course of synthesis and the column
chromatographic separation, the presence of substrates and products
was checked by thin-layer chromatography using silica gel plates
(visualization by UV lamp). The concentration of substrates and products
were determined using UV–VIS spectrometer (Shimadzu-UV-1202,
Kyoto, Japan). The 1H NMR spectra were recorded on 200 MHz Unity
+ spectrometer using heavy water as solvent and TMS (tetramethylsilane)
This enzyme was used for determination of deuterium KIE and
SIE in decomposition of 5′-chloro-L-tryptophan.
Phenylketonuria (PKU)11 is a human genetic disease caused
by a mutation in the gene responsible for coding of L-
phenylalanine dehydrogenase enzyme (PheDH, EC 1.4.1.20),
which converts L-Phe to L-tyrosine. The accumulated L-Phe is as internal standard.
Scheme 3. The reversible enzymatic synthesis/decomposition of L-Trp catalyzed by the enzyme tryptophanase (EC 4.1.99.1).
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M. Kańska et al.
Scheme 4. The enforced metabolic pathways occurring in persons suffering from PKU disease.
Scheme 5. The deamination of 2′-chloro-[(3S)-2H]-L-Phe catalyzed by the enzyme
phenylalanine dehydrogenase (EC 1.4.1.20).
Synthesis and kinetics runs
Scheme 8. Synthesis of [(3R)-3H]-L-Phe, 3.
KIE values for the elimination of ammonia from L-Phe catalyzed by
the enzyme PAL
All isotopomers of L-Phe labeled with deuterium, tritium, and 14C, needed
for kinetics assays, were synthesized according to the methods described
earlier. The isotopomer [1-14C]-L-Phe, 1, (used as internal radiometric
standard) was prepared by the conversion of [1-14C]-CH3COONa in acetic
anhydride, [1-14C]-(CH3O)2O and the followed condensation of the
resultant product with an benzaldehyde, PhCHO, leading to [1-14C]-
cinnamic acid, which was enzymatically converted13 into 1, Scheme 6.
The isotopomer [2-14C]-L-Phe, 2, was obtained by the, partly similar,
combined chemical and enzymatic methods14 using [2-14C]-malonic acid
as a source of label, Scheme 7. Also, the isotopomers [(3R)-3H]-L-Phe, 3,
Scheme 8, and [(3S)-3H]-L-Phe, 4, Scheme 9, were synthesized by the
chemical and enzymatic procedures.15 The doubly labeled [(3S)-2H/3H]-
Scheme 9. Synthesis of [(3S)-3H]-L-Phe, 4, using the enzyme phenylalanine
ammonia lyase (EC 4.3.1.5).
L-Phe, 5, was obtained according to the Scheme 9 by addition of
ammonia to cinnamic acid carried out in fully deuteriated incubation
medium containing tritiated water. The deuteriated [(3R)-2H]-L-Phe, 6,
was synthesized as in Scheme 8 by cleaving benzil by the cyanide ion
dissolved in heavy water and condensing the resulted deuteriated
benzaldehyde, PhC2HO, with malonic acid. The addition of ammonia to
resulted [2-2H]-cinnamic acid, catalyzed by PAL leads to 6. [1H NMR
spectrum of native L-Phe: δ 3.115 (1H, β-HS, d); 3.283 (1H, β-HR, d); 3.983
(1H, α-H, d); 7.325 (2H, 2′and 6′-ArH, m); 7.371 (1H, 4′-ArH, m); 7.423(2H,
3′and 5′ArH). 1H NMR spectrum of 6 – δ 3.117 (1H, β-HS, d); 3.989 (1H,
α-H, t); 7.327 (2H, 2′ and 6′-ArH, m); 7.374 (1H, 4′-ArH, m); 7.425 (2H, 3′
and 5′-ArH)]. The loss of the signal of the β-HR-proton (δ 3.283) in the
spectrum of 6 proves the near 100% incorporation of deuterium in this
position. Also, the isotopomer [(3S)-2H]-L-Phe, 7, was obtained as in
Scheme 9 by the addition of ammonia to cinnamic acid in deuteriated
incubation medium. [1H NMR for 7 – δ 3.285 (1H, β-HR, d); 3.987 (1H, α-
H, t); 7.326 (2H, 2′ and 6′-ArH, m); 7.375 (1H, 4′-ArH, m); 7.426 (2H, 3′and
5′-ArH)]. The disappearance of the signal from β-HR-proton (δ 3.283) in
Scheme 6. Synthesis of [1-14C]-L-Phe, 1 catalyzed by the enzyme phenylalanine
ammonia lyase (EC 4.3.1.5).
Scheme 7. Synthesis of [2-14C]-L-Phe, 2.
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M. Kańska et al.
the spectrum of 7 proves the near 100% incorporation of deuterium in
this position.
Spectrophotometric procedure for KIE determination on KM
(Michaelis constant) and KIE on Vmax (maximum reaction rate) for
[(3R)-2H]-, 6 and [(3S)-2H]-L-Phe, 7. The reaction of ammonia
elimination was carried out in the 6 mL quartz spectrophotometric
cuvette containing the reaction mixture and placed inside measuring
spectrometer’s chamber. The reaction was carried out in 0.2 M boric
buffer (pH 8.7) at 25oC. The progress of reaction was measured on the
basis of the increasing absorbance of cinnamic acid at λ = 290 nm. The
results are given in the Table 2.
Procedure for 14C KIE determination for the 1- and 2-positions of L-
Phe. The kinetic runs of ammonia elimination from L-Phe were carried
out in 25 mL encapped vials containing reaction mixture (labeled
isotopomers of L-Phe, buffer, and enzyme PAL) at 25°C. The
concentration and the specific activity of [1-14C]-, and [2-14C]-L-Phe in
the mixture prepared for kinetics assays were determined as follows:
The concentration of L-Phe in a precise sampled volume of reaction
mixture was determined indirectly by its conversion into cinnamic acid,
catalyzed by enzyme PAL. The subsequent spectroscopic measurement
of absorbance of this product allows to calculate the concentration of
L-Phe (c). The radioactivity (r) of exactly similar sample of reaction mixture
was measured using liquid scintillation counter. Thus, the specific activity
of substrate before the start of reaction – R0 = r/c. In the course of kinetic
assays, the samples with the exact similar volume were taken from the
reaction mixture, and the reaction was quenched by the acidification to
pH ~ 0. The unconverted substrate (L-Phe) and the resultant product
(cinnamic acid) were separated by the extraction with 3 × 10 mL portions
of diethyl ether. The concentration of cinnamic acid was determined
spectroscopically while the radioactivity of L-Phe and cinnamic acid was
measured on scintillation counter. This approach allows to calculate R0,
f (conversion factor), Rs (specific activity of L-Phe at the f), and Rp (specific
activity of cinnamic acid at f). The 14C KIE values for the 1- and 2-positions
of L-Phe, calculated on the basis of formulas of Tong and Yankwich16 are
given in the Table 1.
Procedure for 14C KIE determination in [1-14C]-L-DOPA, 8, in its
decarboxylation catalyzed by tyrosine decarboxylase (EC 4.1.1.25)
This KIE was determined using the tritium ring labeled isotopomer
[2′,5′,6′-3H3]-L-DOPA as the internal radiometric standard, which was
obtained by the acid-catalyzed exchange between tritiated water and
authentic L-DOPA carried out at 50°C as described earlier.17,18 The
assayed substrate 8 was synthesized by the hydroxylation of commercial
[1-14C]-L-tyrosine catalyzed by enzyme tyrosinase according to slightly
modified method,19,20 Scheme 10. In this case of kinetic run, only the
R0, RS, and f parameters were determined. First, the 3H/14C ratio (R0) of
reaction mixture used for kinetics assays (composed of [1-14C]- and
[2′,5′,6′-3H3]-L-DOPA) was measured. The measurements of kinetic
parameters for the calculation of KIE were carried out as follows: the
exact portion of two aforementioned isotopomers of L-DOPA were added
to the 25 mL reaction vial containing 0.1 M phosphate buffer (pH 5.5). The
reaction (carried out at 25°C) started after addition of tyrosine
decarboxylase enzyme and PLP. In the 5-min intervals, the 1 mL samples
were taken and immediately frozen in liquid nitrogen. In the course of
each experiment, six samples were collected, and the degree of
conversion (f) of L-DOPA into dopamine was in the range of 0.15–0.38.
The substrate from product was separated on chromatographic column
(Amberlit IRC-50(H+), 10 × 100 mm) by elution with 0.1 M phosphate
buffer (pH 6.5). The fractions containing radioactivity were collected,
lyophilized, and their radioactivity measured. The Rs and f were
calculated from the radioactivity measurements.
Procedure for H/3H KIE determination in the (3R)-position of L-Phe. For
these kinetic runs as the internal radiometric standard 1 was used. First, for
the prepared reaction mixture, containing the samples of [(3R)-3H]-, and
[1-14C]-L-Phe in the boric buffer (pH 8.7), the total radioactivity of 14C and
the 3H/14C radioactivity ratio (R0) were determined. During the reaction,
the samples were taken at the specified time periods in order to achieve
the degrees of conversion (f) in the range of 2–20%. The elimination
reaction for each taken sample was immediately quenched by the
acidification to pH ~ 0, and cinnamic acid was extracted with diethyl ether,
and its 3H/14C ratio (Rp) was determined. The conversion factor (f) was
calculated by dividing the 14C-radioactivity in the starting sample by the
14C-radioactivity of sample taken in course of kinetics run.
Procedure for deuterium KIE and SIE determination for the
decomposition of 5′-chloro-L-Trp using
a
noncompetitive
spectrophotometric method
5′-chloro-[2-2H]-L-Trp, 9, was synthesized by coupling 5-chloroindole with
S-methyl-L-cysteine catalyzed by the enzyme TPase in fully deuteriated
Procedure for H/3H and 2H/3H KIE determination in the (3S)-
position of L-Phe. For the kinetic runs 1, 3, and 5, that is, the
isotopomers of [1-14C]-, [(3S)-3H]-, and [(3S)-2H/3H]-L-Phe were used.
The procedure for determining some kinetic parameters (R0 and f) of
the reaction was similar, but some modifications should be performed
because of tritium label was loss by cinnamic acid (product) during
the course of reaction. Thus, instead of Rp, the Rr (3H/14C of unreacted
substrate) was determined. Therefore, after aqueous layer extraction,
the remaining unreacted L-Phe was loaded onto chromatographic
column (Amberlite IR-120 H+, 10×100 mm, Aldrich, Poznan, Poland),
washed with water to remove the labile tritium label. Then, L-Phe was
eluted with 0.3 M NH3(aq), concentrated under the reduced pressure,
its 3H/14C ratio (Rr) was determined by liquid scintillation counting,
and KIE was calculated.
incubation medium according to the procedure described earlier21,22
;
see Scheme 11. The product was obtained with 17% chemical yield
and the near 100% deuterium incorporation into the α-position of 9 as
shown by 1H NMR spectrum. [1H NMR for authentic 2′-chloro-L-
tryptophan – δ 3.355 (2H, β-H, m); 4.034 (1H, α-H, q); 7.235 (1H, 6′-ArH,
dd); 7.336(1H, 2′-ArH, s); 7.473 (1H, 7′-ArH, d); 7.731(1H, 4′-ArH, d). 1H
Table 2. Deuterium KIE’s values in the (3R)- and (3S)-
positions for deamination of L-Phe.
Substrate
KIE on Vmax
KIE on Vmax/KM
[(3R)-2H]-L-Phe, 6
[(3S)-2H]-L-Phe, 7
1.031 0.009
1.143 0.012
1.032 0.01
1.134 0.015
Table 1. Tritium and 14C KIE’s values for deamination of
L-Phe.
Substrate
KIE
[1-14C]-L-Phe, 1
1.006 0.008
0.993 0.006
1.055 0.005
1.30 0.02
[2-14C]-L-Phe, 2
[(3R)-3H]-L-Phe, 3
[(3S)-3H]-L-Phe, 4
[(3S)-2H/3H]-L-Phe, 5
Scheme 10. Synthesis of [1-14C]-L-DOPA, 8, catalyzed by the enzyme tyrosinase
(EC 1.14.18.1).
1.08 001
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M. Kańska et al.
Scheme 11. Synthesis of 5′-chloro-[2-2H]-L-Trp, 9, catalyzed by the enzyme tryptophanase (EC 4.1.99.1).
NMR spectrum for 9 – δ 3.229 (2H, β-H, m); 7.239 (1H, 6′-ArH, dd); 7.339
Synthesis of [(4R)-2H]-NADPH, 11, Scheme 12. A sample of 66 mg
(0.5 mmol) of D,L-oxaloacetic acid dissolved in 2H2O was reduced to D,L-
[2-2H]-malic acid with 5 mg (0.12 mmol) of NaB2H4 at 0°C. The procedure
of separation and purification of the obtained D,L-[2-2H]-malic acid was
the same as described earlier26 giving the 53.4 mg (0.3 mmol) sample
of deuteriated product obtained with 60% chem. yield. Then, 2.5 units
of malic enzyme (EC 1.1.1.40) and 15 mg (0.02 mmol) of NADP+ were
added to 15 mg (0.08 mmol) portion of D,L-[2-2H]-malic acid dissolved
in TRIS · HCl buffer. The reaction was carried out for 1 h at 30°C. The
(1H, 2′-ArH, s); 7.479 (1H, 7′-ArH, d); 7.736(1H, 4′-ArH, d)] The loss of signal
from the α-proton of 9 (4.034) indicates near complete incorporation of
deuterium in this position. During the kinetic runs, the progress of 5′-
chloro-L-Trp decomposition was measured indirectly as the substrate
and products, that is, 5-chloroindole and pyruvate, do not adsorb the
photons with energy suitable for the detection with
a
spectrophotometer. For this purpose, to the mixture, an excess of
enzyme lactate dehydrogenase (LDH – EC 1.1.1.27) and NADH were
added. LDH catalyzes the conversion of pyruvate to L-lactate which
oxidizes NADH to NAD+. The formed NADH adsorbs strongly at
λ = 340 nm, whereas NAD+ is invisible. This detailed spectrometric
procedure for measuring kinetic parameters, needed for the calculation
of KIE and SIE, is described elsewhere.23 The values of KIE and SIE are
presented in Table 3.
separation and purification (made by the procedures given earlier27,28
)
gave 9.6 mg (0.011 mmol) of 11 (obtained with 57% chem. yield). The
near 100% deuterium enrichment in the (4R)-position were proved by
1H NMR spectra. In the authentic NADPH, the signals from the (4S)- and
(4R)-protons (δ 2.757, 1HS, and board d), and δ (2.793, 1HR, and broad t)
are compatible with literature data,29 while for obtained 11, only the
signal from (4S)-protons (δ 2.752, 1H4S, and broad d) is observed, while
the signal from the (4R)-protons has disappeared. The 1H NMR spectrum
of authentic NADHP: δ 2.757 (1H, H4S, broad d), 2.793 (1H, H4R, broad
t), 4.038 (2H, d), 4.186 (2H, m), 4.264 (1H, m), 4.375 (1H, m), 5.952 (1H, d),
6.196 (1H, d), 6.918 (1H, s), 8.222 (1H, s), 8.464 (1H, s). The 1H NMR
spectrum of product 11 – δ 2.752 (1H, H4S, broad d), 4.049 (2H, d),
4.180 (2H, m), 4.213 (1H, m), 4.393 (1H, m), 5.952 (1H, d), 6,189 (1H, d),
6.964 (1H, s), 8.225 (1H, s), 8.496 (1H, s).
Procedure for deuterium KIE and SIE determination for the
deamination of 2′-chloro-L-phenylalanine to 2′-chlorophenylpyruvic
acid catalyzed by PheDH
2′-Chloro-[(3S)-2H]-L-Phe ], 10, for this oxidative-deaminative reaction was
obtained by addition of ammonia to 2′-chlorocinnamic acid (Scheme 6)
catalyzed by the enzyme PAL carried out in fully deuteriated incubation
medium as described earlier.15 The product, that is, 10 was obtained with
48% chemical yield and the near 100% deuterium incorporation into the
(3S)-position of 10 , as shown by 1H NMR spectra. [1H NMR for authentic
2′-chloro-L-phenylalanine – δ 3.086 (1H, β-H(S), d); 3.096 (1H, β-H(R), d);
3.705 (1H, α-H, t); 6.908 (1H, 3′-ArH, m); 6.975 (1H, 5′-ArH, m); 7.153 (1H,
6′-ArH, m); 7.312 (1H, 4′-ArH, m). 1H NMR spectrum for 10 δ 3.096 (1H,
β-H (R), s); 3.705 (1Hα, d); 7.142 (1H, 3′-ArH, m); 7.182 (1H, 5′-ArH, m);
7.313 (1H, 6′-ArH, m); 7.363 (1H, 4′-ArH, m)]. In the region of the 1H
NMR spectrum, covering the signals from the protons of the side chain24
of 10, the signal from the (3S)-proton disappears (δ 3.086) indicating the
100% deuterium incorporation in this position. The kinetic runs of
oxidative–deaminative conversion of 10 into 2′-chlorophenylpyruvic acid
were carried out in 0.1 M glycine buffer (pH 10.7) in presence of PheDH
and NAD+. The progress of this reaction was monitored
spectroscopically25 by measuring the increase in absorbance (at
λ = 340 nm) of NADH formed.
Synthesis of [(4S)-2H]-NADPH, 12, Scheme 13. This was carried out
according to the procedure described previously.29 A sample of 15 mg
(0.02 mmol) of NADH+ was reduced by 10 mg (0.055 mmol) of [1-2H]-D-
glucose in the presence of 136 units of enzyme glucose-L-dehydrogenase
(EC 1.1.1.47). As a result, 10.5 mg (0.012 mmol) of 12 were obtained with
62.5% chem. yield and 100% deuterium enrichment at the (4S)-position
verified by 1H NMR spectrum. Only the signal from the (4R)-protons (δ
2.798, H4R, broad t) is observed, while the signal from the (4S)-protons
disappeared. The 1H NMR spectrum of product 12, δ 2.798 (1H, H4R
,
broad t), 4.031 (2H, d), 4.197 (2H, m), 4.259 (1H, m), 4.376 (1H, m),
5.947 (1H, d), 6.188 (1H, d), 6.923 (1H, s),8.218 (1H, s), 8.468 (1H, s).
Noncompetitive spectrophotometric method. For the kinetic runs for
the determination of parameters, needed to calculate the KIE and SIE
values for the reduction of DOPAL to DOPET, the noncompetitive
spectrophotometric method was used. The rate of this reaction, carried
out in phosphate buffer (pH 7.2 or pD 7.6 for protonated or deuteriated
medium, respectively) and catalyzed by enzyme ALDH in the presence
Procedure for deuterium KIE and SIE determination for the (4R)-
and (4S)-positions of NADPH during reduction of DOPAL to DOPET
catalyzed by ALDH
The substrates, that is, [(4R)-2H]-, 11, and [(4S)-2H]-NADPH, 12, for kinetic of [(4R)-2H]- 11, or [(4S)-2H]-NADPH 12, (cofactor) was determined by
runs for the reduction of DOPAL to DOPET were synthesized according to
the measuring at λ = 340 nm the increase of absorbance of NADP+
known procedures.26–29
formed. The calculated KIE and SIE for this reaction are given in Table 4.
Table 3. The deuterium KIE’s and SIE’s values for the 2-position of 5′-chloro-L-Trp for its decomposition catalyzed by TPase.
Substrate
KIE on Vmax
KIE on Vmax/KM
5′-chloro-L-Trp in H2O/ 5′-chloro-[2-2H]-L-Trp, 8, in H2O
1.80 0.65
1.11 0.08
1.87 0.15
1.59 0.11
2
5′-chloro-L-Trp in H2O/ 5′-chloro-[2-2H]-L-Trp, 8, in H2O
SIE on Vmax
2.83 0.19
1.75 0.13
SIE on Vmax/KM
0.68 0.06
0.58 0.03
5′-chloro-L-Trp in H2O/ 5′-chloro-[2-2H]-L-Trp, 8, in H2O
2
5′-chloro-L-Trp in H2O/ 5′-chloro-[2-2H]-L-Trp, 8, in H2O
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M. Kańska et al.
Scheme 12. Synthesis of [(4R)-2H]-NADPH, 11.
Scheme 13. Synthesis of [(4S)-2H]-NADPH, 12, catalyzed by the enzyme glucose 1-dehydrogenase (EC 1.1.1.47).
h
i
Table 4. KIE’s values for oxidation NADPH to NADP+ during
the reduction of DOPAL to DOPET.
R0Þ
ln ð1 ꢀ fÞꢁ
k1
k2
Rs
KIE ¼
¼
(1)
(2)
ln½1 ꢀ fꢂ
ꢀ
ꢁ
Substrate
KIE on Vmax
KIE on Vmax/KM
f ꢁR0
ln 1 ꢀ
lnð1 ꢀRfpÞ
k1
k2
[(4R)-2H]-NADPH, 11
[(4S)-2H]-NADPH, 12,
2.52 0.02
1.46 0.09
2.8 0.2
2.08 0.32
KIE ¼
KIE ¼
¼
R ꢀR
ð
0Þ
p
SIE on Vmax
2.31 0.15
SIE on Vmax/KM
1.27 0.11
ln
R ꢀR
k1
k2
ð
sÞ
p
DOPAL
¼
(3)
R ꢁ R ꢀR
ð
0Þ
s
p
ln
R ꢁ R ꢀR
ð
sÞ
0
p
KIE, kinetic isotope effect; SIE, solvent isotope effect;
NADPH, nicotinamide adenine dinucleotide phosphate;
DOPAL, 3′,4′-dihydroxyphenylacelaldehyde; DOPET, 3′,4′-
dihydroxyphenylethanol.
ꢂ
ꢃ
fꢁ R ꢀR
ð
sÞ
p
1
ln
ꢀ
ð1-fÞ
ð1ꢀfÞꢁRs
k1
k2
ꢂ
ꢃ
KIE ¼
¼
(4)
fꢁ R ꢀR
ð
sÞ
p
1
ln ð1-fÞ ꢀ
ð1ꢀfÞꢁRsꢁRp
where: R0 – starting molar activity of substrate; Rs or Rp – molar
activities of substrate or product, respectively, at given degree
of conversion, f- degree of conversion.
Results and discussion
The kinetic parameters needed to calculate the values of 14C KIE
for the 1- and 2-positions during the deammination of L-Phe to
cinnamic acid catalyzed by enzyme PAL, and carried out at 30o,
were determined using the competitive radiometric method30;
see Scheme 1. The KIE = k1/k2 (reaction rates with 12C and 14C,
respectively) was calculated using Yankwich–Tong Equations16
14C- KIE values were determined at low degrees of conversion
(f – from 0.1 to 0.23) and using three parameters selected from
the set of four (R0, Rs, Rp, and f) and four equations to obtain
the compatible results indicating that during the kinetics
measurements the systematic errors were avoided. The
(1)–(4) and the kinetic data measured in the course of five calculated values of 14C KIE values for the 1- and 2-position of
independent experimental runs.
L-Phe are near 1 – (1.006 0.005 and 1. 008 0.006). This means
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J. Label Compd. Radiopharm 2016

M. Kańska et al.
that there is no KIE in this position, and so these two in the early steps of elimination of ammonia, which was
isotopomers can be used as internal radiometric standards to confirmed by semi empiric calculation. For the confirmation of
investigate the tritium KIE in the aforementioned reaction as these hypotheses, the investigation of carbon KIE at the ortho
the 14C-label is distant from the center of reaction and can not position of L-Phe is needed.
affect its rate.
The lack of large isotope effects in ammonia elimination
Tritium KIE values were determined for low conversion of from L-Phe catalyzed by PAL proves that the mechanism is
substrate (f = 0.1 – 0.25) and using [1-14C]-L-Phe as internal complicated and it is difficult to identify the rate determining
radiometric standard. In these cases, KIE values were calculated the step of this reaction. Perhaps, more than one stage has a
using only one from Equations (1) to (4) and the parameters from decisive influence on the overall rate of reaction. The attention
five independent kinetics runs.
must be paid to the following facts: the size of tritium KIE in
For the (3R)-position of [(3R)-3H]-L-Phe, 3, the secondary the (3S)- and (3R)-position,(1.3 and 1.08, respectively), however,
H/T KIE was calculated using the isotopic ratios 14C/3H of both being small, differ from each other. The (3S)-position
substrate (R0) and cinnamic acid (Rp) isolated after reaction (where hydrogen is abstracted in the process of elimination)
and Equation (2).
is more isotopically sensitive. KIE in the (3S)-position may be
For the (3S)-position of [(3S)-3H]-L-Phe, 4, the primary H/T KIE the result of specific interaction of (3S)-proton with the
was determined using isotopic ratios 14C/3H of substrate before enzyme, while KIE in the (3R)-position may be associated with
(R0) and isolated after reaction (Rs), and Equation (1).
a change of hybridizing with sp3 to sp2. It is likely that the
For the (3S)-position of [(3S)-2H/3H]-L-Phe, 5, the D/T KIE was internal isotope effect at this position is substantially greater
determined using isotopic ratios 14C/3H of substrate before (R0) than that observed and may be suppressed by other processes
and cinnamic acid isolated after reaction (Rp), and Equation (2). of a similar rate.
The summarized KIE, values are listed in Table 1.
The determined 14C KIE for the 2-position of L-Phe (to which
The kinetic parameters for calculation of deuterium KIE’s, that amino group is attached) is about 1 (no effect), which indicates
is, KIE on Vmax and KIE on Vmax/KM, for the reaction presented in that the leaving amino group does not decide of the reaction
Scheme 1, were determined using the noncompetitive rate. The lack of KIE excludes the two-step (E2) and carbocation
spectrophotometric method.30 The absorbance measurements (E1) mechanisms. It is most likely the carboanion (equivalent
of cinnamic acid allow to determine Vmax and calculation KM E1cB) mechanism; that is, first, a proton is abstracted, and later,
according to Michaelis Equation (5).
the amino group is leaving. Having considered that, a DHA
(cofactor) electrophilic attack on the ring and formation a
positive charge on C1-ring carbon, proposed by Shuster and
Retey,6 seem likely.
ꢄ
Vmax
ꢅ
KM ¼ S
v
ꢀ 1
(5)
14C KIE for [1-14C]-L-DOPA in its decarboxylation catalyzed by
tyrosine decarboxylase (EC 4.1.1.25) (Scheme 2) was determined
using the tritium ring labeled isotopomer [2′,5′,6′-3H3]-L-DOPA as
the internal radiometric standard. In this case, only 14C/3H ratios
of starting substrate (R0), substrate, isolated after conversion (Rs)
and f parameter were determined. KIE equal to 1.18 0.01 was
calculated using Equation (1). Here, the rarely found high value
of 14C KIE proves that Cα–CCOOH bond breakage occurs in the rate
determining step of this enzymatic reaction.
where v is the reaction rate at substrate concentration S, Vmax is a
maximum reaction rate, and KM is a Michaelis constant. KM and
Vmax are interrelated, and approximately, KM is a measure of
strength of enzyme-substrate binding, and Vmax is a measure of
the rate of reaction under conditions given.
The deuterium KIE’s for (3R)- and (3S)-positions of L-Phe are
given in Table 2.
Despite of the numerous literature data, the mechanism of
deamination of L-Phe catalyzed by the enzyme PAL is still unclear
and has gained much attention for long time. The mechanism
proposed by Hanson and Havier5 assumes that dehydroalanine
(DHA) – the rare prosthetic group forming essential part of PAL
and the important factor for catalysis – acts as the electrophile
forming a covalently bonded ‘enzyme-intermediate’ complex
with quaternary nitrogen of L-Phe. Therefore, this complex
enhances the leaving ability of the amino group and withdraws
negative charge from the hydrogen at β-carbon, so it becomes
sufficiently acidic to be abstracted by a base. The alternative
mechanism postulated by Schuster and Retey6 assumes that in
the first step of this reaction, a Friedel–Crafts type of electrophilic
attack of DHA on the aromatic ring occurs, followed by hydrogen
abstraction. To carry out ammonia elimination from L-Phe
(Scheme 1), two events are necessary: firstly, the activation of
hydrogen at β-carbon making it capable to leave, and secondly,
the quarternization of Phe-nitrogen and thus facilitating the C-
N-rupture. The Hanson and Havir’s mechanism5 assumes that
DHA plays both roles simultaneously, in contrary to the Shuster
and Retey’s mechanism,6 where DHA activates a hydrogen, while
protonation of Phe is a separate step occurring upon. Also the
data published7 of H/T KIE at ortho position of ring of L-Phe
provides a strong argument for the Friedel–Crafts reaction type
The deuterium KIE’s and SIE’s to the 2-position of side chain
for 5′-chloro-[2-2H]-L-Trp, 8, during its decomposition to 5-indole
and pyruvate (similar as shown in Scheme 3) were calculated
using Equation (5) and kinetic parameters determined as stated
in the Experimental Section are listed in Table 3.
The earlier reported31 isotope effects for the 2-position for
decomposition of [2-2H]-L-Trp (listed in the succeeding section)
carried out in the same conditions differ insignificantly from
those given in Table 3.
• KIE on Vmax = 2.1 0.1 and 2.1 0.13 (in H2O and 2H2O,
respectively)
2
• KIE on Vmax/KM = 2.10 014 and 1.45 0.13 (in H2O and H2O,
respectively)
• SIE on Vmax = 1.87 0.17 and 1.25 0.17 (in H2O and 2H2O,
respectively)
2
• SIE on Vmax/KM = 1.07 013 and 1.14 0.05 (in H2O and H2O,
respectively)
This means that substitution of hydrogen atom by the
chlorine one does not affect the reaction of degradation of
chloroderivative of L-Trp in comparison to [2-2H]-L-Trp.
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M. Kańska et al.
In the course of oxidative deamination of 2′-chloro-[(3S)-2H]-L- of 5′-chloro-L-Trp by TPase, as wells as the biotransformations
Phe, 8, Scheme 5, the deuterium KIE’s and SIE’s for the (3S)- catalyzed by dehydrogenases, that is, oxidative deamination
position were determined as described in Experimental Section. of 2′-chloro-L-Phe by PhDH and the conversion of DOPAL to
The kinetic data needed for isotope effects calculation were DOPET by ALDH using deuteriated in the (4R)- or (4S)-positions
obtained using Equation (5). The numerical values of deuterium NADPH as cofactor. These KIE and SIE values may be useful to
in this position are: For the KIE’s on Vmax and the KIE’s on Vmax
/
elucidate some mechanism details of the investigated
K
M – 1.20 0.09 and 1.35 0.12, respectively; for the SIE’s on Vmax enzymatic reactions.
and the SIE’s on Vmax/KM
– 1.46 0.12 and 2.28 0.13,
respectively.
References
Earlier published32 or not published yet isotope effects
(listed in the succeeding sectionn) for the 3-position for
oxidative deamination of [(3S)-2H]-L-Phe carried out in the
same conditions are
[1] J. T. McFarland, In Enzyme Mechanism from Isotope Effects (Ed.: P. F.
Cook), CRS, Boca Raton, Ann Harbor: Boston, London, 1991,
151–265.
[2] D. H. Jones, Phytochemistry 1984, 23, 1349.
[3] C. N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T. M. S.
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Microbiol. 1981, 42, 773.
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[6] B. Shuster, J. Rètey, Proc. Natl. Acad. Sci. 1995, 92, 8433.
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Biochem. Biophys. 1999, 370, 216.
[8] G. H. Sloane-Stanley, Biochem. J. 1949, 44, 373.
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[10] W. A. Wood, I. C. Gunsalus, W. W. Umbreit, J. Biol. Chem. 1947, 170,
313.
• For the KIE’s on Vmax and the KIE’s on Vmax/KM – 1.16 0.12 and
1.01 0.09, respectively
• For the SIE’s on Vmax and the SIE’s on Vmax/KM – 1.67 0.11 and
1.91 0.13, respectively.
As can be seen from earlier data, the values of isotope effects
are almost identical (with limits of experimental error), which
means that ring substitution of hydrogen atom by chlorine does
not affect the oxidative deamination of 2′-chloro-[(3S)-2H]-L-Phe
in comparison to [(3S)-2H]-L-Phe.
Small or close to 1 (no effect) the deuterium KIE and SIE values,
observed in the decomposition of 5′-chloro-[2-2H]-L-Trp and 2′-
chloro-[(3S)-2H]-L-Phe for the α- and β-protons, strongly suggest
that a proton abstraction from these positions in not the rate
determining step of the reactions mentioned.
[11] C. R. Scriver, Hum. Mutat. 2007, 28, 831.
[12] G. A. Mitchell, M. Grompe, M. Lambert, R. M. Tanguay, In The
Metabolic and Molecular Bases of Inherited Disease (Eds.: C. R. Scriever,
A. L. Beaudet, W. S. Sly, D. Valle), 8th edn, Vol II, McGraw-Hill: New
York, 2001, 1777–1785.
[13] J. Jemielity, M. Kańska, R. Kański, Isotop. Environ. Health Stud. 1998,
34, 335.
The kinetic parameters for KIE and SIE values for the enzymatic
reduction of DOPAL to DOPET in the presence of NADPH and
catalyzed by enzyme ALDH (Scheme 2) were determined as
described in Experimental Section and calculated using Equation
(5). The SIE on Vmax = 2.31 0.15 and the SIE on Vmax /KM = 1.27
0.11 are obtained for the reduction of DOPAL to DOPET. The
KIE values for the oxidation of NADPH (reducing species) to
NADP+ for this reaction are listed in Table 4.
[14] W. Augustyniak, J. Bukowski, J. Jemielity, R. Kański, M. Kańska,
J. Radioanal. Nucl. Chem. 2001, 247, 371.
[15] J. Jemielity, R. Kanski, M. Kanska, J. Label. Comp. Radiopharm. 2001,
44, 295.
[16] J. T. Tong, P. E. Yankwich, J. Phys. Chem. 1957, 61, 540.
[17] M. Pająk, M. Kańska, J. Radioanal. Nucl. Chem. 2009, 279, 455.
[18] W. Byszewska, M. Kańska, J. Radioanal. Nucl. Chem. 2014, 299, 1373.
[19] M. Kozłowska, R. Kański, M. Kańska, J. Label. Compd. Radiopharm.
2005, 48, 235.
[20] M. Pająk, M. Kańska, J. Radioanal. Nucl. Chem. 2009, 281, 365.
[21] E. Boroda, R. Kanski, M. Kańska, J. Label. Compd. Radiopharm. 2003,
46, 691.
The higher value of SIE on Vmax than SIE on Vmax/KM (Table 4) is
affiliated with the conformational changes in the structure of
ALDH occurring during the cofactor binding with the enzyme [22] M. Pająk, K. Pałka, E. Winnicka, M. Kańska, J. Label. Compd.
Radiopharm. 2016, 59, 4.
(Vmax is very sensitive to this changes). The published
[23] E. Winnicka, P. Dabrowski, T. Winek, M. Kańska, Isotop. Environ. Health
crystallographic data33 show that ALDH’s conformational
changes occur as a result of the side amino acids group rotation,
participating in the binding of NADPH.
Stud. 2010, 46, 225.
[24] K. R. Hanson, R. H. Wightman, J. Stauton, A. R. Battersby, J. Chem. Soc.
Chem. Commun. 1971, 185.
[25] M. Kanska, K. Skowera, J. Label. Comp. Radiopharm. 2010, 53, 406.
[26] M. Lowenstein, Methods Enzymol. 1963, 6, 871.
[27] C. Cummings, D. Ryerson, D. P. Ballou, C. Walsh, Biochemistry 1982,
21, 1141.
[28] S. Suye, S. Yokohama, Enzyme Microbiol. Technol. 1985, 7, 418.
[29] G. Ottolina, S. Riva, G. Carrea, B. Danielli, A. F. Buckmann, Biochim.
Biophys. Acta 1989, 998, 173.
[30] D. W. Parkin, In Enzyme Mechanism from Isotope Effects (Ed.: P. F.
Cook), CRS, Boca Raton, Ann Harbor: Boston, London, 1991,
269–288.
[31] E. Boroda, R. Kański, M. Kańska, Ann. Pol. Chem. Soc. 2004, 3, 96.
[32] M. Kanska, K. Skowera, J. Label. Comp. Radiopharm. 2010, 53, 443.
[33] O. Keifeld, A. Frenkel, O. Bogin, M. Eisenstein, V. Brumfeld, Y. Burstein,
I. Sagi, Biochemistry 2000, 39, 7702.
Moreover, the higher values of deuterium KIE on Vmax and KIE
on Vmax/KM for [(4R)-2H]- than [(4S)-2H]-NADPH (Table 4) suggest
that during the DOPAL to DOPET reduction, the ALDH enzyme
catalyzes the proton removal from the (4R)-position.34
Conclusion
This research presents some new data on the isotopic effects in
enzymatic reactions. The numerical values of deuterium,
tritium, and 14C KIE and SIE values have been determined for
the following reactions, catalyzed by the enzymes from lyases
group: the deamination of L-Phe by PAL, the decarboxylation
of L-DOPA by tyrosine decarboxylase, and the decomposition
[34] V. Valera, M. Fung, A. N. Wessler, W. R. Richards, Biochim. Biophys. Res.
Commun. 1987, 148, 515.
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