4.6 Article

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

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 36, Pages 34505-34516

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M303359200

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Funding

  1. NIGMS NIH HHS [GM 63477] Funding Source: Medline

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Cloned 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 (K-i = 45 nM), suggesting that the successive C-methylation of the Delta(24) bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [H-2(3)-methyl] AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on V-CH3/V-CD3 close to unity. [25-H-2]24(28)-Methylenecycloartanol, [28E-H-2]24(28)-methylenelanosterol, and [28Z-H-2]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-H-3(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 Delta(24) bond.

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