Cane D E, Pargellis C
Arch Biochem Biophys. 1987 May 1;254(2):421-9. doi: 10.1016/0003-9861(87)90120-2.
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.
戊塔烯合酶是一种催化法呢基焦磷酸环化形成倍半萜烃戊塔烯的酶，已通过阴离子交换、羟基磷灰石和凝胶过滤色谱法相结合的方法，从链霉菌UC5319的上清液部分中部分纯化出来。通过凝胶过滤色谱法估计部分纯化的合酶的分子量为57,000，并且环化酶活性与非变性聚丙烯酰胺圆盘凝胶电泳可见的八条主要蛋白带中的一条相关。法呢基焦磷酸的Km为0.77±0.21微摩尔，部分纯化的合酶的Vmax为每小时每毫克蛋白287±21纳摩尔戊塔烯。环化酶活性需要二价金属阳离子的存在。虽然Mg2+或Mn2+都可以使用，但Mn2+在浓度高于2.5毫摩尔时具有抑制作用。不需要其他辅因子。在约10微摩尔的浓度下，产物戊塔烯和无机焦磷酸均未显示出对环化酶活性的显著抑制作用，但两者的组合导致法呢基焦磷酸的表观Km增加了约七倍，这表明两种产物都可以在活性位点协同结合，以竞争性抑制戊塔烯合酶。
