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.