Elżbieta Winnicka

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′-2H]-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′-2H]-l-tryptophan fully deuteriated in the 5′ -position.

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 2H2O, catalysed by enzyme tryptophanase (EC 4.1.99.1). The positional deuterium enrichment of the resulting tryptophan derivatives was controlled using 1H 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, β-F-l-alanine, synthesized from β-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.

Isotope effects in the hydroxylation of l -tyrosine catalyzed by tyrosinase

The reaction mechanism of the enzymatic hydroxylation of l-tyrosine (l-Tyr) to l-DOPA catalyzed by enzyme tyrosinase (EC 1.14.18.1) were investigated using the kinetic (KIE) and solvent (SIE) isotope effect methods. The values of deuterium KIE’s and SIE’s for [2′,6′-2H2]-, and [3′,5′-2H2]-l-Tyr were obtained using a non-competitive spectrophotometric method but values of H/T and D/T KIE’s for the 3′- and 5′-positions of [3′,5′-3H2]-, and [3′,5′-2H/3H]-l-Tyr were determined by competitive method with [1-14C]-l-Tyr as internal radioactive standard.

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)-2H]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 [2-2H]-l-tryptophan. [(1R)-2H]tryptamine was obtained by enzymatic decarboxylation of l-tryptophan in the fully deuterated medium.