Biosynthesis of phytosterols: Kinetic mechanism for the enzymatic C-methylation of sterols

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dc.creatorNes, W. David (TTU)
dc.creatorSong, Zhihong (TTU)
dc.creatorDennis, Allen L. (TTU)
dc.creatorZhou, Wenxu (TTU)
dc.creatorNam, Jaewook (TTU)
dc.creatorMiller, Matthew B. (TTU)
dc.date.accessioned2023-09-01T16:40:32Z
dc.date.available2023-09-01T16:40:32Z
dc.date.issued2003
dc.descriptioncc-by
dc.description.abstractCloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C 1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-L-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nM), suggesting that the successive C-methylation of the Δ24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on V CH3/VCD3 close to unity. [25- 2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylenelanosterol were prepared and paired with AdoMet or [methyl-3H 3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Δ24 bond.
dc.identifier.citationNes, W.D., Song, Z., Dennis, A.L., Zhou, W., Nam, J., & Miller, M.B.. 2003. Biosynthesis of phytosterols: Kinetic mechanism for the enzymatic C-methylation of sterols. Journal of Biological Chemistry, 278(36). https://doi.org/10.1074/jbc.M303359200
dc.identifier.urihttps://doi.org/10.1074/jbc.M303359200
dc.identifier.urihttps://hdl.handle.net/2346/95956
dc.language.isoeng
dc.titleBiosynthesis of phytosterols: Kinetic mechanism for the enzymatic C-methylation of sterols
dc.typeArticle

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