title: Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogues of 2, 3-oxidosqualene.
abstract: Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evaluated as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius. In these analogues, sulfur replaces carbons at C-6, C-10, C-14, C-18, or C-19 of OS. Each analogue was a submicromolar inhibitor of SHC with IC50 values ranging from 60 to 570 nM. Enzyme inhibition kinetic analysis was performed using homogeneous recombinant A. acidocaldarius SHC. While analogues 9 (S-14, Ki = 109 nM, kinact = 0.058 min-1) and 11 (S-19, Ki = 83 nM, kinact = 0.054 min-1) were time-dependent inhibitors of SHC, analogues 7 (S-6, Ki = 127 nM) and 8 (S-10, Ki = 971 nM) showed no time dependency with SHC. Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (Ki = 31 nM, kinact = 0.071 min-1). Kinetic analysis for the five analogues with purified rat liver OSLC was conducted to compare the vertebrate and prokaryotic enzymes. Affinity labeling experiments, using either [17-3H]10 or [22-3H]10 with crude and with pure recombinant SHC, clearly showed specific labeling. A single major radioactive band at 72 kDa on SDS-PAGE indicated that irreversible covalent modification of SHC had occurred. These results suggest that the presence of sulfur at C-18 of OS can interrupt the cyclization and that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.
authors.affiliation: Department of Medicinal Chemistry, The University of Utah, Salt Lake City 84112-5820, USA.
authors.forename: Y F
authors.initials: YF
authors.lastname: Zheng
authors.forename: I
authors.initials: I
authors.lastname: Abe
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001406:Bacillaceae / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats; D013185:Squalene / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
chemical_list: C002821:2,3-oxidosqualene; D000345:Affinity Labels; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D013185:Squalene; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; C021924:lanosterol synthase