Abstract
Pentalenene synthase, an enzyme which catalyzes the cyclization of farnesyl pyrophosphate to the sesquiterpene hydrocarbon pentalenene, has been partially purified from the supernatant fraction of Streptomyces UC5319 by a combination of anion-exchange, hydroxylapatite, and gel-filtration chromatography. The molecular weight of the partially purified synthase was estimated by gel filtration chromatography to be 57,000 and the cyclase activity was shown to be associated with a major protein band among eight visible by nondenaturing polyacrylamide disc gel electrophoresis. The K m for farnesyl pyrophosphate was 0.77 ± 0.21 μ m and the V max for the partially purified synthase was 287 ± 21 nmol of pentalenene/mg protein per hour. Cyclase activity required the presence of a divalent metal cation. Although either Mg 2+ or Mn 2+ could be used, Mn 2+ was inhibitory at concentrations above 2.5 m m. No other cofactors were required. Whereas neither product, pentalenene nor inorganic pyrophosphate, showed significant inhibition of cyclase activity at concentrations of ca 10 μ m, the combination of the two resulted in an approximate sevenfold increase in the apparent K m for farnesyl pyrophosphate, suggesting that both products can bind cooperatively at the active site to inhibit pentalenene synthase competitively.
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