Partial purification and characterization of pentalenene synthase.
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 Km for farnesyl pyrophosphate was 0.77 +/- 0.21 microM and the Vmax 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 Mg2+ or Mn2+ could be used, Mn2+ was inhibitory at concentrations above 2.5 mM. No other cofactors were required. Whereas neither product, pentalenene nor inorganic pyrophosphate, showed significant inhibition of cyclase activity at concentrations of ca 10 microM, the combination of the two resulted in an approximate sevenfold increase in the apparent Km for farnesyl pyrophosphate, suggesting that both products can bind cooperatively at the active site to inhibit pentalenene synthase competitively.
D002413:Cations, Divalent; D002845:Chromatography; D003517:Cyclopentanes / Q000494:pharmacology; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019753:Intramolecular Lyases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D012717:Sesquiterpenes; D013302:Streptomyces / Q000201:enzymology*
D002413:Cations, Divalent; D003517:Cyclopentanes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D008345:Manganese; C024646:pentalenene; C004808:farnesyl pyrophosphate; D007535:Isomerases; D019753:Intramolecular Lyases; C048278:farnesylpyrophosphate cyclase; D008274:Magnesium