Изучение влияния концевых структурных модификаций на функции цитохромов Р450 микросом млекопитающих

Выводы:

1. Получены вектора для экспрессии вариантов цитохромов Р450 1А2 человека и 2В4 кролика в штаммах Е. coli и S. cerevisiae. Модифицированные цитохромы несли: на N-конце — введенный сайт протеазы Ха на границе пролин богатого района (ПБР) и А-спирали либо домен GST, на С-концеолигогистидиновые а.о. либо домен интеина. Оптимизированы условия экспрессии и очистки мутантных белков.

2. CYP1A2 и CYP2B4 с С-концевыми олигогистидиновыми остатками не отличаются по своим энзиматическим свойствам от природных белков. В то же время рекомбинантный фермент 2В4(Д2−27) с удаленным N-концевым сегментом обладает пониженной каталитической активностью. Показана принципиальная невозможность применения метода аффинной очистки С-концевых гибридов Р450 с интеином вследствие инактивации гемопротеи-на при индукции автопротеолиза гибрида.

3. Рекомбинантный аналог CYP1A2 с введенным сайтом узнавания фактора Ха на границе ПБР и А-спирали обладает измененными энзиматическими свойствами по сравнению с немодифицированным ферментом. При этом возникает новая субстратная специфичность в отношении 7-ПОРФ и 7-БОРФ и негиперболическая зависимость между скоростью реакции О-деметилирования и концентрацией субстрата 7-МОРФ.

4. Прокариотическая НАДФН-цитохром Р450 редуктаза BMR из B. megaterium способна выступать в качестве эффективного редокс-партнера CYP1A2 человека.

Работа поддержана грантом INTAS 96−1549 и ИНКО-КОПЕРНИКУС №.

ERB3512PL965070 (контракт № ERBIC15CT960810).

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232. Я крайне благодарен моему научному руководителю К. Г. Скрябину, принявшему самое непосредственное участие в моей научной судьбе.

233. Необходимо отдельно выразить самую глубокую благодарность H.A. Пе-тушковой, научившей меня работать с препаратами Р450, посвятившей в многочисленные тонкости их физико-химических измерений.

234. Настоящая работа посвящается моим дорогим родителям, без которых эта работа не была бы выполнена, Г. М. Щукиной и A.A. Жгуну старшему.