Isoprene formation in a rat liver cytosolic fraction is shown to be increased 146-fold by acid treatment. This acid catalysis is dependent upon prior incubation of the cytosolic fraction with DL-mevalonate and is stimulated when the incubation also contains ATP. Formation of isoprene proceeds linearly through 5 h of acid treatment and is nearly complete at 10 h. These results suggest that the acid-catalyzed isoprene formation arises from the decomposition of dimethylallyl pyrophosphate via a carbonium ion mechanism. Chemical model studies using 3-methyl-2-buten-1-ol and 3-methyl-3-buten-1-ol (the alcohols corresponding to dimethylallyl pyrophosphate and isopentenyl pyrophosphate, respectively) confirm this hypothesis. At a pH less than or equal to 1, an 85% decomposition of 3-methyl-2-buten-1-ol to isoprene occurred after 24 h, while 3% of 3-methyl-3-buten-1-ol was converted to isoprene under identical conditions and time. It is concluded that the predominant immediate precursor of isoprene is dimethylallyl pyrophosphate and at low pH the ultimate fate of dimethylallyl pyrophosphate is complete conversion to isoprene. These conclusions have important biochemical and methodological implications.

The invitro biosynthesis of isoprene from DL-mevalonate in the cytosolic fraction of rat liver is described. Evidence is provided suggesting a non-enzymatic formation of isoprene from isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate. Furthermore, the data establish an alternate fate of the mevalonate carbon skeleton providing the first evidence that breath isoprene is linked to cholesterol biosynthesis.

The activity of rat lung epoxide hydrolase (epoxide hydrolase, EC 3.3.2.3) was studied using two lipid epoxides which can be isolated from lung tissue. These epoxides displayed different Km,app and hydration rates. Methyl cis-9,10-epoxystearate was hydrated 20-times more rapidly than cholest-5α,6α-epoxy-3β-ol. The Km for the lung microsomal enzyme was variable and dependent on the microsome concentration in the medium. A soluble epoxide hydrolase was also detected in both lung and liver. This enzyme appears similar to the microsomal enzyme in its activity toward methyl epoxystearate. The measurement activities for liver microsomal epoxide hydrolase were over 8-times those for lung microsomes; activity against cholesterol epoxide was 40-times greater for liver. In spite of the slow rates measured with cholesterol epoxide in lung preparations, this compound was an effective competitive inhibitor against methyl epoxystearate over a wide concentration range. This suggests that cholesterol epoxide readily binds to epoxide hydrolase and is an effective competitive inhibitor against a much more actively metabolized substrate, methyl epoxystearate. Such circumstances indicate that cholesterol epoxide binds with a high degree of nonproductivity to lung microsomal epoxide hydrolase. This attribute of lung epoxide hydrolase may relate to the relatively high concentrations of cholesterol epoxide found in lung tissue.

A soluble cytochrome P-450 monooxygenase system from Bacillusmegaterium ATCC 14581, previously shown to catalyze the monohydroxylation of longchain unsubstituted fatty acids, has now been found to convert 9-D-hydroxystearate to a mixture of ω-1, ω-2, ω-3 and ω-4 dihydroxystearate isomers. 9-D-Hydroxystearate has a significantly higher affinity than stearate for the enzyme and is a strong competitive inhibitor of palmitate hydroxylation. These results suggest that the enzyme surface has a non-hydrophobic, sterically-permissive binding region between the methyl-group and carboxyl-group binding sites that interacts with polar substituents near the middle of the substrate chain.