5600235568

I Konferencja Naukowa Enzymos Enzymy w nauce i przemyśle Wystąpienia Gości Honorowych_

meTHF. Ponadto, atomy C(5) i C(6) cząsteczki inhibitora wskazywały na hybrydyzację sp3, sugerując redukcję C(5), brak było wskazań uwolnienia protonu z C(5), a katalityczna Cysl89 związana była kowalencyjnie z atomem C(6). W zgodzie z tym, N⁴-OH-dCMP pozostawał związany z enzymem po denaturacji. Tak więc inhibitor wydaje się uczestniczyć jako samobójczy substrat w abortywnej reakcji katalizowanej przez enzym, prowadzącej do redukcji pirymidynowego C(5) i utlenienia THF do DHF.

Mechanism of thymidylate synthase-catalyzed reaction inhibition by N⁴-OH-dCMP in view of crystallographic studies

Thymidylate synthase (TS; EC 2.1.1.45), a target in chemotherapy, catalyzes dUMP methylation to dTMP associated by conversion of N⁵’¹⁰-methylenetetrahydrofolate (meTHF) to dihydrofolate (DHF). A substrate analogue, N⁴-hydroxy-dCMP (N⁴-OH-dCMP), similar to 5-fluoro-dUMP (FdUMP), is a competitive with respect to dUMP, and time- and meTHF-dependent TS inhibitor. However, unlike [5-³H]dUMP, it does not release ³H from the uracil ring C(5) while replacing the substrate in the reaction mixture. Surprisingly, only the anti-imino isomer appears to be the active inhibitor form.

Structures were solved, based on crystals formed by mouse TS (mTS) protein in the presence of meTHF and either FdUMP (1.55A resolution) or N⁴-OH-dCMP (1.17A resolution). While FdUMP-containing structure revealed a covalently bound enzyme-FdUMP-meTHF ternary complex, as expected, N⁴-OH-dCMP-containing structure showed the enzyme's ternary complex with N⁴-OH-dCMP (the anti imino isomer) and non-covalently bound DHF, instead of expected meTHF. Moreover, both C(5) and C(6) inhibitor atoms showed the sp3 hybridization, suggesting C(5) reduction, with no indication of the C(5) proton release and with the catalytic Cysl89 covalently bound to the C(6) atom. In accord, N⁴-OH-dCMP remained bound to the enzyme following denaturation. Thus the inhibitor appears to participate as a suicide substrate in an abortive enzyme-catalyzed reaction, resulting in the pyrimidine C(5) reduction and THF oxidation to DHF.

20