Inhibition Kinetics and Affinity Labeling of Bacterial Squalene:Hopene Cyclase by Thia-Substituted Analogues of 2,3-Oxidosqualene † Yi Feng Zheng, Ikuro Abe, and Glenn D. Prestwich* Department of Medicinal Chemistry, The UniVersity of Utah, 30 South 2000 East, Room 201, Salt Lake City, Utah 84112-5820 ReceiVed NoVember 4, 1997; ReVised Manuscript ReceiVed February 11, 1998 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 IC 50 values ranging from 60 to 570 nM. Enzyme inhibition 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 ) were 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. Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (K i ) 31 nM, k inact ) 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- 3 H]10 or [22- 3 H]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. The enzymatic cyclizations of squalene 1 and oxi- dosqualene 2 (OS) 1 convert acyclic polyalkenes to polycyclic products that are precursors of critical membrane constituents (1). Bacterial squalene-hopene cyclase (SHC) (EC 5.4.99.7) is believed to bind 1 in an all prechair conformation and then catalyze the sequential formation of five new C-C bonds through a series of carbocationic intermediates leading to a hopanyl C-22 carbocation 3. Either H-29 proton elimination or addition of H 2 O at 3 results in release of the final products hop-22(29)-ene (4) and hopan-22-ol (5), respectively (Scheme 1A) (2). In contrast, the eukaryotic oxidosqualene:lanosterol cyclase (OSLC) (EC 5.4.99.7) folds 2 in chair-boat-chair conformation and mediates the cycliza- tion of 2 through enzyme-constrained carbocationic inter- mediates to the protosteryl (C-20) cation. Backbone rear- rangement of the cation leads to lanosterol 6 (Scheme 1B). These two cyclizations both generate and stabilize carboca- tionic intermediates as key steps in the catalytic process. Bacterial squalene cyclases will also accept epoxides as substrates and can catalyze the cyclization of 2 into penta- cyclic triterpenes by initiating oxirane ring opening rather than by protonating the terminal double bond (3-6). For instance, bacterial SHC can cyclize (3R) and (3S)-OS to 3R- hydroxyhopene and 3-hydroxyhopene, respectively (3, 4). The molecular details of how these cyclases fold their substrates and orchestrate specific cation-induced polyolefinic ring formation remains to be elucidated. Recent advances in molecular and structural biology have spurred mechanistic studies of these enzymes. Several bacterial SHCs have been purified (7-9), cloned, and functionally expressed (10-12). SHCs are membrane- associated 70-75 kDa proteins. Sequence comparison of bacterial SHCs and eukaryotic OSLCs have showed 17- 27% overall amino acid identity, with greater identity in the C-terminal region. In addition, eight repeats of a highly conserved R-helix turn motif rich in aromatic amino acids (the QW motif) (13, 14) were recognized as key structural elements. Site-directed mutagenesis experiments on OSLC and SHC have demonstrated that D-456 in yeast OSLC was essential for catalytic function (15, 16), while the homologous residues D-376 and D-377 in A. acidocaldarius SHC were crucial for enzyme activity (17). Most recently, a 2.9 Å resolution X-ray crystal structure of Alicyclobacillus aci- docaldarius SHC was reported (18, 19). SHC is a ho- modimeric enzyme, and each unit consists of 631 amino acids with molecular mass of 71 569 Da. Two R-helical domains create the hydrophobic active site of the enzyme, identified in a large central cavity by using the bound † This work was supported by the National Institutes of Health (Grant GM 44836 to G.D.P.) and The University of Utah. * To whom correspondence should be addressed: Phone: 801 585 9051. Fax: 801 585 9053. E-mail: gprestwich@deans.pharm.utah.edu. 1 Abbreviations: OS, 2,3-oxidosqualene; SHC, squalene: hopene cyclase; OSLC, oxidosqualene:lanosterol cyclase; 29-MOS, (3S)-29- methylidene-2,3-oxidosqualene; S-19, 19-thia-18-dehydro-(R, S)-2,3- oxidosqualene; S-18, 18-thia-19-dehydro-(R,S)-2,3-oxidosqualene; S-14, 14-thia-15-dehydro-(R,S)-2,3-oxidosqualene; S-10, 10-thia-11-dehydro- (R,S)-2,3-oxidosqualene; S-6, 6-thia-7-dehydro-(R,S)-2,3-oxidosqualene; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; AMO 1618; 5-hydroxylcarvacryl trimethylammonium chloride 1-piperidine carboxylate; DodMe 2NO, dodecyldimethylamine-N-oxide; DodMe3NBr, dodecyltrimethylammonium bromide; Ro48-8071, [4′- (6-allyl-methyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl- methanone fumarate; BIBX79, trans-N-(4-chlorobenzoyl)-N-methyl- (4-dimethylaminomethylphenyl)-cyclohexylamine. 5981 Biochemistry 1998, 37, 5981-5987 S0006-2960(97)02734-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/03/1998