Impact of Effectors on the Catalytic Activity of Galactonolactone Oxidase from Trypanosoma cruzi

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Abstract

The influence of the structure of the effectors, 1,4-benzoquinone, coenzymes Q and their structural analogues, on the activity of galactonolactone oxidase from Trypanosoma cruzi (TcGAL) and the homologous enzyme L-galactono-1,4-lactone dehydrogenase from Arabidopsis thaliana (AtGALDH) was studied. Using two forms of AtGALDH, natural (dehydrogenase) and mutant (exhibiting oxidase activity), the role of 1,4-benzoquinone and its analogs as electron acceptors of AtGALDH and TcGAL was revealed. It has been established that compounds containing methoxy groups are more effective electron acceptors for TcGAL (coenzyme Q0, 2,6-dimethoxy-1,4-benzoquinone) compared to compounds without OCH3 groups (2,5-dihydroxy-1,4-benzoquinone). Using 2,6-dimethoxy-1,4-benzoquinone as an electron acceptor, an approach to the spectrophotometric measurement of TcGAL activity by changes in the absorption of the electron acceptor in the absence of additional components (a dye that becomes colorless when interacting with the reaction product, ascorbate) is proposed. The results obtained allow for a more targeted search for TcGAL inhibitors, which can be considered as the basis for the development of selective drugs against Chagas disease caused by T. cruzi.

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About the authors

A. A. Chudin

Lomonosov Moscow State University

Author for correspondence.
Email: andrew_18@inbox.ru

Chemical Department

Russian Federation, Leninskie gory 1/3, Moscow, 119991

E. V. Kudryashova

Lomonosov Moscow State University

Email: Helenakoudriachova@yandex.ru

Chemical Department

Russian Federation, Leninskie gory 1/3, Moscow, 119991

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Supplementary files

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2. Fig. 1. Catalytic cycle of the enzymes AtGALDH and TcGAL.

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3. Fig. 2. Structures of 1,4-benzoquinone (a) and its analogs: coenzyme Q0 (b), coenzyme Q1 (c), 2,6-dimethoxy-1,4-benzoquinone (d), 2,5-hydroxy-1,4-benzoquinone (d) and thymoquinone (e).

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4. Fig. 3. Maximum activity of the natural form of AtGALDH in aqueous (sodium phosphate buffer) (a) and micellar (0.1 M AOT in n-octane, W₀ = 22) (b) media depending on the structure of EA in the presence of the dye DCPIP. Concentrations: substrate (1 mM), DCPIP (120 μM), AtGALDH (6 nM). The concentrations of EA at which the maximum enzyme activity was observed are listed in Table 1.

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5. Fig. 4. Maximum activity of the mutant form of AtGALDH in aqueous (sodium phosphate buffer, pH 8.8 for CoQ0 and 2,6-dimethoxy-BC and pH 7.8 for CoQ1 and BC) (a) and micellar (0.1 M AOT in n-octane, pH 8.8 for CoQ0 and 2,6-dimethoxy-BC and pH 7.8 for CoQ1 and BC, W₀ = 22) (b) media depending on the structure of EA in combination with the dye DCPIP. Concentrations: substrate (1 mM), DCPIP (120 μM), AtGALDH (6 nM). Activity without the addition of EA is indicated for comparison.

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6. Fig. 5. Dependence of the maximum activity of TcGAL in micellar medium (0.1 M AOT in n-octane, pH 8.8, W0 = 22) on the structure of the effector (electron acceptor) in combination with the dye DCPIP. The concentrations of the substrate (1 mM), DCPIP (120 μM) and TcGAL (34 nM) were maintained constant. For comparison, the activity of TcGAL in the presence of 120 μM PMS is shown according to previously published data [3]. The concentrations of effectors (electron acceptors) at which the maximum enzyme activity was observed: 2,5-dihydroxy-BC (360 μM), thymoquinone (200 μM), CoQ0 (0.72 μM), 2,6-dimethoxy-BC (24 μM). The concentration of FMS in control measurements is 120 μM.

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7. Fig. 6. (a) – Spectra of the oxidized and reduced forms of 24 μM 2,6-dimethoxy-BC in sodium phosphate buffer, pH 8.8); (b) – spectra of the oxidized and reduced forms of 24 μM 2,6-dimethoxy-BC in a micellar medium (0.1 M AOT in n-octane, W₀ = 22, pH 8.8); (c) – spectra of the reaction mixture (1 mM D-arabinono-1,4-lactone, 24 μM 2,6-dimethoxy-BC) in a micellar medium (0.1 M AOT in n-octane, W₀ = 22) before and after the reaction (addition of 34 nM TcGAL); (g) – dependence of TcGAL activity on the concentration of 2,6-dimethoxy-BC (without dye) and on the concentration of CoQ0 (in combination with 120 μM DCPIP), the baseline is the activity of TcGAL with a combination of 120 μM FMS and 120 μM DCPIP.

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8. Fig. 7. (a) – Scheme of target reaction inhibition; (b) – comparison of the inhibitory effect of allyltetramethoxybenzene and allylbenzene for dye-containing systems (combination of DCPIP dye and PMS or Q0 as EA) and a system without dye (2,6-dimethoxy-BC as EA). Concentrations of substances: 120 μM DCPIP, 1 mM AR (substrate), 34 nM TcGAL, 24 μM 2,6-dimethoxy-BC, 120 μM PMS and 0.72 μM Q0, 200 μM allyltetramethoxybenzene and 200 μM allylbenzene. Medium: 0.1 M AOT in n-octane, W₀ = 22.

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