Studies on the biosynthesis of cholesterol. XII. Synthesis of allyl pyrophosphates from mevalonate and their conversion into squalene with liver enzymes

The biosynthesis of allyl pyrophosphates (the pyrophosphates of dimethylallyl alcohol, geraniol, and of farnesol) from dl-mevalonate-2-C14 and from (-)5-phosphomevalonate-2-C14 with soluble liver enzymes in the presence of ATP and Mg++ is described. The allyl pyrophosphates were partially purified and their properties studied. They are unstable below pH 5 and cleave into inorganic pyrophosphate (identified by the use of purified yeast inorganic pyrophosphatase) and allylic alcohols. The alcohol components were identified by gas-liquid radiochromatography after hydrolysis of the pyrophosphates by prostatic, intestinal, microsomal, and snake venom phosphatases. Farnesyl pyrophosphate, which was the principal product among the allyl pyrophosphates, was shown to be the precursor of squalene in the liver enzyme system just as was reported for yeast. The enzyme or enzymes responsible for the conversion of farnesyl pyrophosphate into squalene are attached to microsomal particles and need either TPNH or DPNH and a divalent cation (Mg++, Mn++, or Co++) as cofactors. Slightly better yields of squalene were obtained with TPNH than with DPNH. The squalene synthesizing system was strongly inhibited by p-chloromercuribenzoate and N-ethyl maleimide, but was not affected by iodoacetamide; the optimal pH was 7.4. Addition of soluble enzymes to this system reduced the yield of squalene and caused some conversion of the allyl pyrophosphates into carboxylic acids.