Thiol modification of diacylglycerol kinase: dependence upon site membranedisposition and reagent hydrophobicity

Reaction rates were determined between disulfide reagents of varying hydrop hobicity and single-cysteine mutants of diacylglycerol kinase, an integral membrane protein, Polar reagents reacted most rapidly with surface-exposed sites. However, a very non-polar reagent also reacted more rapidly with exp osed cysteines than with membrane sites, Moreover, this non-polar reagent u sually reacted more slowly with membrane sites than did more polar reagents . These results are consistent with the notion that disulfide exchange reac tions involving buried cysteines of diacylglycerol kinase are very slow in the membrane interior, such that the competing rates of reactions which occ ur when normally buried cysteine sites make motional excursions to hydrated regions of the interface can be significant. (C) 2000 Federation of Europe an Biochemical Societies.