PARTIAL-PURIFICATION AND CHARACTERIZATION OF PENTALENENE SYNTHASE
PARTIAL-PURIFICATION AND CHARACTERIZATION OF PENTALENENE SYNTHASE
复制标题
DOI：
10.1016/0003-9861(87)90120-2
复制
发表时间：
1987-05-01
影响因子：
3.9
通讯作者：
PARGELLIS, C
中科院分区：
文献类型：
--
作者：
CANE, DE;PARGELLIS, C
并环戊二烯合酶是一种催化焦磷酸法呢酯环化为倍半萜烃并环戊二烯的酶，已通过阴离子交换、羟基磷灰石和凝胶过滤色谱的组合从链霉菌UC 5319的上清液级分中部分纯化。用凝胶过滤层析法估计部分纯化的合成酶的分子量为57，000，用非变性聚丙烯酰胺圆盘凝胶电泳法显示环化酶活性与8条可见蛋白带中的一条主要蛋白带有关。焦磷酸法呢酯的Km为0.77 ±。0.21部分纯化的合酶的Vmax为287 ± 0.5 μ M。每小时21 nmol并环戊二烯/mg蛋白质。环化酶活性需要二价金属阳离子的存在。虽然可以使用Mg 2+或Mn 2+，但Mn 2+在浓度高于2.5 mM时具有抑制作用。不需要其他辅因子。尽管在约10 μ M的浓度下，产物，并环戊二烯和无机焦磷酸都没有显示出对环化酶活性的显著抑制，但两者的组合导致法尼基焦磷酸的表观Km增加了约7倍，表明两种产物可以在活性位点协同结合以竞争性地抑制并环戊二烯合酶。
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 .mu.M 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 .mu.M, 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.