Studies on the purification and characterization of Sterol Methyl Transferase from Glycine max

dc.creatorDennis, Allen L.
dc.description.abstractC-Methylations of the sterol side-chain at carbon-24 (phytosterols) serve as markers of diversity between the kingdoms in biology. The consecutive C-methylation steps are important in the biosynthesis phytosterols plants. S-Adenosyl-L-Methionine to A Sterol Methyltransferase (SMT), the enzyme responsible for catalyzing the Cmethylation reaction of substrate acceptor molecules in Glycine max, was subcloned into erg6 yeast and BL21 (DE3) bacterial cells. Kinetic parameters, inclucding substrate specificity and product distribution, of the SMT enzyme were characterized. SMT was found to catalyze both C-methylation steps at the same active site; however, the first Cmethylation was catalyzed at a much higher effeciency. Substrate specificity studies with 17 structurally modified sterols indicated that cycloartenol was the preferred substrate for the first C-methylation and 24(28)-methylene-lophenol was the preferred substrate for the second C-methylation reaction. SMT transformed cycloartenol into a single sterol product, where as 24(28)-methyl-lophenol gave rise to three products. Studies involving isotopically labeled substrates, stereospecific inhibition of SMT and inhibition by transition state analogs are consistant with the steric-electric plug model, which proposes a noncovalent p-face C-methylation mechanism. Kinetic mechanism studies indicated SMT follows a sequential ordered mechanism.
dc.publisherTexas Tech Universityen_US
dc.subjectMethyltransferases -- Purificationen_US
dc.titleStudies on the purification and characterization of Sterol Methyl Transferase from Glycine max