| O-Farnesyl (1-thio)diphosphate {dollar}rm (FPPalpha S){dollar} and O-presqualene (1-thio)diphosphate {dollar}rm (PSPPalpha S){dollar} were synthesized and used as alternate substrates for a truncated soluble form of recombinant yeast squalene synthase. The phosphorothioate analogues of FPP and PSPP were designed to trap reactive carbocationic intermediates by a thiono-tholo rearrangement between phosphorothioate anion and the intimate ion pair generated during catalysis. Product studies showed that squalene synthase catalyzed the hydrolysis of {dollar}rm FPPalpha S{dollar} to O-farnesyl thiophosphate (FTP) without a concomitant thiono-thiolo rearrangement. The rate of hydrolysis of {dollar}rm FPPalpha S{dollar} was much slower than the squalene synthase catalyzed conversion of FPP to squalene. Studies of the divalent metal dependent hydrolysis of {dollar}rm FPPalpha S{dollar} indicated that the hydrolysis was fourfold faster in the presence of {dollar}rm Mnsp{lcub}2+{rcub}{dollar} than in the presence of {dollar}rm Mgsp{lcub}2+{rcub}. FPPalpha S{dollar} was a competitive inhibitor of squalene synthase against FPP with {dollar}rm Ksb{lcub}I{rcub} = 47 mu{dollar}M.; Product analysis study indicated that {dollar}rm PSPPalpha S{dollar} was an alternative substrate for squalene synthase. In the presence of NADPH, the enzyme catalyzed the synthesis of squalene, 10-hydroxybotrycoccene (a {dollar}1spprime{dollar}-3 isoprenoid), 12-hydroxysqualene (a 1{dollar}spprime{dollar}-1 isoprenoid), and 12,13-cis-dehydrosqualene (a 1{dollar}spprime{dollar}-1 isoprenoid) in 88%, 11%, and 1% yields, respectively. In the absence of NADPH, only 10-hydroxybotrycoccene, 12-hydroxysqualene, and 12,13-cis-dehydrosqualene were formed at rates similar to those observed in the presence NADPH.; The structure of the alcohols is consistent with trapping carbocationic intermediates by water during the enzymatic rearrangement of presqualene diphosphate to squalene. |
