Showing papers on "Lanosterol synthase published in 1997"

Studies on the Substrate Binding Segments and Catalytic Action of Lanosterol Synthase. Affinity Labeling with Carbocations Derived from Mechanism-Based Analogs of 2,3-Oxidosqualene and Site-Directed Mutagenesis Probes

Abstract: Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible, time-dependent inhibitors of the enzyme lanosterol synthase, were found to attach covalently within the 231−236 (yeast number...

Methodology for the Preparation of Pure Recombinant S. cerevisiae Lanosterol Synthase Using a Baculovirus Expression System. Evidence That Oxirane Cleavage and A-Ring Formation Are Concerted in the Biosynthesis of Lanosterol from 2,3-Oxidosqualene

Abstract: Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured Vmax/KM ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the...

Synthesis and inhibition studies of sulfur-substituted squalene oxide analogues as mechanism-based inhibitors of 2,3-oxidosqualene-lanosterol cyclase

Abstract: The synthesis and biological evaluation of three new sulfur-substituted oxidosqualene (OS) analogues (1-3) are presented. In these analogues, C-11, C-15, or C-18 in the OS skeleton was replaced by sulfur. The sulfur position in the OS skeleton was chosen to disrupt one or more key processes involved in cyclization: (a) the folding of the B-ring into a boat conformation, (b) the anti-Markovnikov cyclization leading to the C-ring, or (c) the formation of the D-ring during the lanosterol biosynthesis. Enzyme inhibition kinetics using homogeneous mammalian oxidosqualene cyclases (OSC) were also examined for the previously reported S-19 analogue 4. The four analogues were potent inhibitors of mammalian OSCs (IC50 = 0.05-2.3 microM for pig and rat liver OSC) and fungal cell-free Candida albicans OSC (submicromolar IC50 values). In particular, the S-18 analogue 3 showed the most potent inhibition toward the rat liver enzyme (IC50 = 50 nM) and showed potent, selective inhibition against the fungal enzyme (IC50 = 0.22 nM, 10-fold more potent than the S-19 analogue 4). Thus, 3 is the most potent OSC inhibitor known to date. The Ki values ranged from 0.5 to 4.5 microM for pig OSC, with 3 and 4 showing about 10-fold higher potency for rat liver OSC. Interestingly, the S-18 analogue 3 showed time-dependent irreversible inhibition with homogeneous pig liver OSC (kinact = 0.06 min-1) but not with rat OSC.