Active site mapping and substrate channeling in the sterol methyltransferase pathway

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dc.creatorNes, W. David (TTU)
dc.creatorMarshall, Julie A. (TTU)
dc.creatorJia, Zhonghua (TTU)
dc.creatorJaradat, Tahhan T. (TTU)
dc.creatorSong, Zhihong (TTU)
dc.creatorJayasimha, Pruthvi (TTU)
dc.date.accessioned2023-09-01T16:40:34Z
dc.date.available2023-09-01T16:40:34Z
dc.date.issued2002
dc.descriptioncc-by
dc.description.abstractSterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified from Escherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor [3-3H]26,27-dehydrozymosterol (26,27-DHZ). A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18 reverse phase high performance liquid chromatography. Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling. Equilibration studies indicated that zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereas S-adenosylmethionine was bound only to the native SMT, Kd of about 2 μM. Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26′)-trienol as well as 26-homocholesta-8(9),26(26′)-3β,24β-dienol, and in the case of D79L and E82L mutants, zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol. The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of 1H and 13C NMR techniques. A Km of 15 μM, Kcat of 8 × 10-4 s-1, and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents. Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the zymosterol high energy intermediate as a consequence of less than perfect control. Alternatively, the gain in enzyme function resulting from the production of a Δ25(27)-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.
dc.identifier.citationNes, W.D., Marshall, J.A., Jia, Z., Jaradat, T.T., Song, Z., & Jayasimha, P.. 2002. Active site mapping and substrate channeling in the sterol methyltransferase pathway. Journal of Biological Chemistry, 277(45). https://doi.org/10.1074/jbc.M204223200
dc.identifier.urihttps://doi.org/10.1074/jbc.M204223200
dc.identifier.urihttps://hdl.handle.net/2346/95958
dc.language.isoeng
dc.titleActive site mapping and substrate channeling in the sterol methyltransferase pathway
dc.typeArticle

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