Isotope effects in the tyrosinase catalysed hydroxylation of l-tyrosine methyl derivatives.

To investigate isotope effects in the hydroxylation of [3',5'-2H2]-α-methyl- and [3',5'-2H2]-N-methyl-l-tyrosine, they were synthesised using acid catalysed isotope exchange at high temperature. The kinetic and solvent deuterium isotope effects on Vmax and Vmax/Km parameters of tyrosinase in its action on methylated derivatives of l-tyrosine were determined using the non-competitive spectrophotometric method. Lineweaver-Burk plots were used to consider the inhibition type of O-methyl-l-tyrosine, revealing that it is an uncompetitive inhibitor of tyrosinase.

The chemo- enzymatic synthesis of labeled l-amino acids and some of their derivatives.

This review compiles the combined chemical and enzymatic synthesis of aromatic l-amino acids (l-phenylalanine, l-tyrosine, l-DOPA, l-tryptophan, and their derivatives and precursors) specifically labeled with carbon and hydrogen isotopes, which were elaborated in our research group by the past 20 years. These compounds could be then employed to characterize the mechanisms of enzymatic reactions via kinetic and solvent isotope effects methods.

Enzymatic synthesis of methyl derivatives of L-tryptophan selectively labeled with hydrogen isotopes.

We report the enzymatic synthesis of the derivatives of L-tryptophan methylated in indole moiety and labeled with deuterium and tritium in the 2-position of side chain. For kinetic studies twelve isotopomers, i.e., 1'-methyl-[2-2H]-, 1'-methyl-[2-3H]-, 1'-methyl-[2-2H/3H]-, 2'-methyl-[2-2H]-, 2'-methyl-[2-3H]-, 2'-methyl-[2-2H/3H]-, 5'-methyl-[2-2H]-, 5'-methyl-[2-3H]-, 5'-methyl-[2-2H/3H]-, 7'-methyl-[2-2H]-, 7'-methyl-[2-3H]-, and 7'-methyl-[2-2H/3H]-L-tryptophan are obtained by the enzymatic coupling of the appropriate methylated indole moiety with S-methyl-L-cysteine catalyzed by the enzyme tryptophanase.

Enzymatic syntheses of 3'-halotyramines, selectively 2H- and 3H-labeled in the side chain.

3'-Halotyramines, stereo-selectively labeled with deuterium and tritium, in the (1R) or (1S) position were synthesized using enzymatic methods. The isotopomers labeled in the (1R) position were synthesized by enzymatic decarboxylation of an appropriate 3'-halo-L-tyrosine in deuterated or tritiated incubation medium. The isotopomers labeled in the (1S) position were synthesized by decarboxylation of an appropriate 3'-halo-L-tyrosine, labeled with deuterium or tritium in the α-position of the side chain.

Isotope effects in mechanistic studies of l-tyrosine halogen derivatives hydroxylation catalyzed by tyrosinase.

The kinetic (KIE) and solvent (SIE) isotope effect methods were used to investigate the mechanism of enzymatic hydroxylation of halogenated derivatives of l-tyrosine to l-DOPA catalyzed by the enzyme tyrosinase (EC 1.14.18.1). The values of deuterium KIE and SIE were obtained using the non-competitive method with spectrophotometric measurements. The Lineweaver-Burk plots were used for determination of the inhibition mode of 3'-iodo-l-tyrosine. Based upon kinetic effects values the mechanism of action of enzyme tyrosinase was proposed.

Isotopic effects in mechanistic studies of biotransformations of fluorine derivatives of L-alanine catalysed by L-alanine dehydrogenase.

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 2H2O - 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.

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

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.

Synthesis of deuterium-labelled halogen derivatives of L-tryptophan catalysed by tryptophanase.

The isotopomers of halogen derivatives of l-tryptophan (l-Trp) (4'-F-, 7'-F-, 5'-Cl- and 7'-Br-l-Trp), specifically labelled with deuterium in α-position of the side chain, were obtained by enzymatic coupling of the corresponding halogenated derivatives of indole with S-methyl-l-cysteine in (2)H2O, catalysed by enzyme tryptophanase (EC 4.1.99.1). The positional deuterium enrichment of the resulting tryptophan derivatives was controlled using (1)H NMR. In accordance with the mechanism of the lyase reaction, a 100% deuterium labelling was observed in the α-position; the chemical yields were between 23 and 51%. Furthermore, ß-F-l-alanine, synthesized from ß-F-pyruvic acid by the l-alanine dehydrogenase reaction, has been tested as a coupling agent to obtain the halogenated deuterium-labelled derivatives of l-Trp. The chemical yield (∼30%) corresponded to that as observed with S-methyl-l-cysteine but the deuterium label was only 63%, probably due to the use of a not completely deuterated incubation medium.

Syntheses of halogen derivatives of L-tryptophan, L-tyrosine and L-phenylalanine labeled with hydrogen isotopes.

Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3)H]-, 5'-bromo-[2-(2)H/(3)H]-, 5'-fluoro-[2-(3)H]-5'-fluoro-[2-(2)H/(3)H]-, 6'-fluoro-[2-(3)H]-, 6'-fluoro-[2-(2)H/(3)H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3)H]-, 3'-fluoro-[2-(2)H/(3)H]-, 3'-chloro-[2-(3)H]-, and 3'-chloro-[2-(2)H/(3)H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3)H]-, 2'-fluoro-[(3S)-(2)H/(3) H]-, 2'-chloro-[(3S)-(3)H]-, 2'-chloro-[(3S)-(2)H/(3)H]-, 4'-chloro-[(3S)-(3)H]-, and 4'-chloro-[(3S)-(2)H/(3)H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2O) with addition of HTO were used.

Isotope effects in the enzymatic oxidation of tryptamine to 3-indolyl-acetaldehyde.

The reaction mechanisms of the enzymatic deamination of tryptamine catalysed by the enzyme monoamine oxidase (MAO, EC 1.4.3.4) were investigated using the kinetic isotope effect and solvent isotope effect methods. The numerical values of these deuterium effects in the (1S) and (1R) positions of tryptamine were determined using the non-competitive spectrophotometry. The deuterium-labelled isotopologue [(1S)-(2)H]tryptamine was obtained in two steps by enzymatic coupling of indole with S-methyl-l-cysteine in a deuterated medium followed by enzymatic decarboxylation of the resulting [2-(2)H]-l-tryptophan. [(1R)-(2)H]tryptamine was obtained by enzymatic decarboxylation of l-tryptophan in the fully deuterated medium.

Enzymatic synthesis of tryptamine and its halogen derivatives selectively labeled with hydrogen isotopes.

Nine isotopomers of tryptamine and its halogen derivatives, labeled with deuterium, tritium in side chain, i.e., [(1R)-2H]-, [(1R)-3H]-, 5-F-[(1R)-2H]-, 5-F-[(1R)-3H]-, 5-Br-[(1R)-2H]-, double labeled [(1R)-2H/3H]-, 5-F-[(1R)-2H/3H]-, and ring labeled [4-2H]-, and [5-2H]-tryptamine, were obtained by enzymatic decarboxylation of l-Trp and its appropriate derivatives in deuteriated or tritiated media, respectively. Intermediates: [5'-2H]-l-Trp used for further decarboxylation was synthesized by enzymatic coupling of [5-2H]-indole with S-methyl-l-cysteine, and [4'-2H]-l-Trp was obtained by isotope exchange 1H/2H of the authentic l-Trp dissolved in heavy water induced by UV-irradiation. Doubly labeled [(1R)-2H/3H]- and 5-F-[(1R)-2H/3H]-tryptamine were obtain by decarboxylation of l-Trp or [5'-F]-l-Trp carried out in 2H3HO incubation medium.

Studies on enzymatic oxidation of 3',4'-dihydroxy-l-phenylalanine to dopachrome using kinetic isotope effect methods.

We report the studies on the mechanism of oxidation of 3',4'-dihydroxy-l-phenylalanine (l-DOPA) to neurotoxic dopachrome catalyzed by enzyme horseradish peroxidase (EC 1.11.1.7) using the kinetic (KIE), and solvent (SIE), isotope effect methods. For kinetic studies two specifically deuterated isotopomers: [2',5',6'-2H3]-l-DOPA was synthesized by the acid catalyzed isotopic exchange between native l-DOPA and heavy water, and [5'-2H]-l-DOPA was synthesized in two step reaction. The first step involved acid catalyzed isotopic exchange between l-tyrosine and deuterated water and resulting product [3',5'-2H2]-l-tyrosine was hydroxylated by enzyme tyrosinase (EC 1.14.18.1). The values of deuterium KIEs and SIE's in the enzymatic oxidation of l-DOPA and its isotopomers are determined using non-competitive spectrophotometric method. The measured values were: KIE on Vmax (1.1 and 2.2) and KIE on Vmax/KM (1.7 and 3.2) for [2',5',6'-2H3]-l-DOPA and [5'-2H]-l-DOPA, respectively, while the corresponding values of SIE were: SIE on Vmax (2.1, 2.4, and 2.1) and SIE on Vmax/KM (1.3. 1.6, and 1.1) for l-DOPA, [2',5',6'-2H3]-l-DOPA, and [5'-2H]-l-DOPA, respectively. The size of KIE and SIE, typical for secondary isotope effects indicate that both the solvent and presence of deuterium at the 2'-, 5', and 6'-positions of l-DOPA has the little impact on the enzymatic oxidation of this compound.

Mechanistic studies of reactions catalysed by diamine oxidase using isotope effects.

Diamine oxidase (DAO), the enzyme that is responsible for amine biodegradation in animals, plants and humans, catalyses the biotransformation of amines such as histamine (HA), putrescine, 1-phenylethylamine, tyrosine, tryptamine, serotonine and spermine. The kinetic and solvent isotope effects (SIEs) were applied to study the mechanism of the biotransformation using HA and its methylderivatives. The SIE for the biotransformation of HA, N(τ)-methylhistamine and N(π)-methylhistamine was found to be 3.58, 2.22 and 5.70 on Vmax, and 1.58, 1.06 and 1.14 on Vmax/KM, respectively. On the other hand, the kinetic isotope effect for oxidation of stereospecifically deuterium-labelled [(α R)-(2)H]-N(τ)-methylhistamine and [(α R)-(2)H]-N(π)-methylhistamine was 0.69 and 0.62 on Vmax, and 15.06 and 7.50 on Vmax/K(M), respectively. These results demonstrate that DAO catalyses amine biotransformation by stereospecifically cleaving the αC-H bond in the pro-S position. Moreover, the oxidation of amine to aldehyde involves several transition states, including hybridisation change from sp(3) (Schiff base) to sp(2) (imine), then back again to sp(3) to give a final product with hybridisation sp(2) (aldehyde).

Kinetic and solvent deuterium isotope effects in the oxidation of putrescine catalysed by enzyme diamine oxidase.

In this study, the kinetic isotope effects and solvent isotope effects in the reaction of the deamination of [(1R)-(2)H ] putrescine--catalysed by enzyme diamine oxidase (EC 1.4.3.6)--were determined using a non-competitive spectroscopic method. Putrescine, stereospecifically labelled with deuterium, was obtained by enzymatic decarboxylation of l-ornithine that was carried out in a fully deuteriated incubation medium.

The influence of cyclomaltooligosaccharides (cyclodextrins) on the enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase.

Cyclomaltohexaose (α-cyclodextrin) and cyclomaltoheptaose (ß-cyclodextrin) as well as their four methyl ether derivatives, that is, hexakis(2,3-di-O-methyl)cyclomaltohexaose, hexakis(2,3,6-tri-O-methyl)cyclomaltohexaose, heptakis(2,3-di-O-methyl)cyclomaltoheptaose, and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose were investigated as the additives in the course of enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase. Only a few of those additives behaved like classical inhibitors of the enzymatic reaction under investigation because the values of the Michaelis constants that were obtained, as well as the maximum velocity values depended mostly atypically on the concentrations of those additives. In most cases cyclodextrins caused mixed inhibition, both competitive and noncompetitive, but they also acted as activators for selected concentrations. This atypical behaviour of cyclodextrins is caused by three different and independent effects. The inhibitory effect of cyclodextrins is connected with the decrease of substrate concentration and unfavourable influence on the flexibility of the enzyme molecules. On the other hand, the activating effect is connected with the decrease of product concentration (the product is an inhibitor of the enzymatic reaction under investigation). All these effects are caused by the ability of the cyclodextrins to form inclusion complexes.

The kinetic and solvent deuterium isotope effects in the 4- and 5-positions of the indole ring on the enzymatic decomposition of L-tryptophan.

The kinetic and solvent deuterium isotope effects in the 4- and 5-positions of the indole ring on the enzymatic decomposition of l-tryptophan catalysed by the enzyme TPase (EC. 4.1.99.1) were determined. The isotope effects were investigated by the non-competitive method using [4'-(2)H]-l-tryptophan, which was enriched in deuterium in 70% in the 4-position. The numerical values of isotope effects for 100% enrichment in deuterated label in that position were calculated by approximation. Those same isotope effects were determined for [5'-(2)H]-l-tryptophan fully deuteriated in the 5' -position.

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of L-tryptophan by L-tryptophan indole-lyase.

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2'-methoxyethyl)]-alpha-cyclodextrin; heksakis(2,3-di-O-methyl)-alpha-cyclodextrin; heptakis(2,3-di-O-methyl)-beta-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-alpha-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-beta-cyclodextrin; heksakis[2,3-di-O-(2'-methoxyethyl)]-alpha-cyclodextrin and heptakis[2,3-di-O-(2'-methoxyethyl)]-beta-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-alpha-cyclodextrin and (2-hydroxypropyl)-beta-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of L-tryptophan by L-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and L-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.