Squalene synthase: steady-state, pre-steady-state, and isotope-trapping studies.
Squalene synthase catalyzes two consecutive reactions in sterol biosynthesis-the condensation of two molecules of farnesyl diphosphate (FPP) to form the cyclopropylcarbinyl intermediate presqualene diphosphate (PSPP) and the subsequent rearrangement and reduction of PSPP to form squalene. Steady-state and pre-steady-state kinetic studies, in combination with isotope-trapping experiments of enzyme.substrate complexes, indicate that two molecules of FPP add to the enzyme before NADPH and that PSPP is converted directly to squalene without dissociating from the enzyme under normal catalytic conditions. In addition, formation of PSPP or a prior conformational change in squalene synthase is the rate-limiting step for synthesis of squalene from FPP via PSPP in the presence of NADPH and for synthesis of PSPP in the absence of NADPH. Squalene synthase is inhibited at high concentrations of FPP. Inhibition is specific for the formation of squalene, but not PSPP, and is competitive with respect to NADPH. In addition, the binding of either NADPH or a third, nonreacting molecule of FPP stimulates the rate of PSPP formation. A kinetic mechanism is proposed to account for these observations.
Radisky
E S
ES
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Poulter
C D
CD
D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012717:Sesquiterpenes; D013185:Squalene / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium
D002250:Carbon Radioisotopes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D014316:Tritium; C017672:presqualene pyrophosphate; D009249:NADP; C004808:farnesyl pyrophosphate; D013185:Squalene; D006859:Hydrogen; D013186:Farnesyl-Diphosphate Farnesyltransferase