Mechanism of squalene biosynthesis: evidence against the involvement of free nerolidyl pyrophosphate

Several mechanisms that utilize farnesyl pyrophosphate and nerolidyl pyrophosphate as condensing substrates have been postulated for the asymmetric condensation reaction in squalene biosynthesis. Although there is ample evidence that farnesyl pyrophosphate is a substrate for this reaction, there has been no information concerning the role of nerolidyl pyrophosphate. We have made the following observations that demonstrate that nerolidyl pyrophosphate cannot be a free intermediate in squalene biosynthesis. (a) There is no significant interconversion of farnesyl pyrophosphate and nerolidyl pyrophosphate in a squalene-synthesizing system from yeast. (b) Nerolidyl-1-(3)H(2) pyrophosphate is not converted to squalene in the presence or absence of farnesyl pyrophosphate. (c) The addition of unlabeled nerolidyl pyrophosphate to incubation mixtures does not alter the relative loss of alpha-hydrogens from farnesyl pyrophosphate during its conversion to squalene. The synthesis of nerolidyl-1-(3)H(2) pyrophosphate is described. Chromatographic methods for the separation of pyrophosphate esters of triprenols and terpenols are included.