Active site mapping and substrate channeling in the sterol methyltransferase pathway.
Sterol 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, K(d) of about 2 microm. 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')-3beta,24beta-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 (1)H and (13)C NMR techniques. A K(m) of 15 microm, K(cat) of 8 x 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 delta(25(27))-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA. wdavid.nes@ttu.edu
W David
WD
Nes
Julie A
JA
Marshall
Zhonghua
Z
Jia
Tahhan T
TT
Jaradat
Zhihong
Z
Song
Pruthvi
P
Jayasimha
D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D005658:Fungi / Q000201:enzymology; D007700:Kinetics; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000600:Amino Acids, Dicarboxylic; D017931:DNA Primers; D010446:Peptide Fragments; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D007930:Leucine