INHIBITION-KINETICS AND AFFINITY LABELING OF BACTERIAL SQUALENE-HOPENE CYCLASE BY THIA-SUBSTITUTED ANALOGS OF 2,3-OXIDOSQUALENE

Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evalua ted as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacill us 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 in hibitor of SHC with IC50 values ranging from 60 to 570 nM. Enzyme inhi bition kinetic analysis was performed using homogeneous recombinant A. acidocaldarius SHC. While analogues 9 (S-14, K-i = 109 nM, k(inact) = 0.058 min(-1)) and 11 (S-19, K-i = 83 nM, k(inact) = 0.054 min(-1)) w ere time-dependent inhibitors of SHC, analogues 7 (S-6, K-i = 127 nM) and 8 (S-10, K-i = 971 nM) showed no time dependency with SHC. Analogu e 10 (S-18) was the most potent inhibitor and showed time-dependent ir reversible inhibition (K-i 31 nM, k(inact) = 0.071 min(-1)). Kinetic a nalysis for the five analogues with purified rat liver OSLC was conduc ted to compare the vertebrate and prokaryotic enzymes. Affinity labeli ng experiments, using either [17-H-3]10 or [22-H-3]10 with crude and w ith pure recombinant SHC, clearly showed specific labeling. A single m ajor radioactive band at 72 kDa on SDS-PAGE indicated that irreversibl e covalent modification of SHC had occurred. These results suggest tha t the presence of sulfur at C-18 of OS can interrupt the cyclization a nd that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.