IDENTIFICATION OF THE ACTIVE-SITE OF VERTEBRATE OXIDOSQUALENE CYCLASE
IDENTIFICATION OF THE ACTIVE-SITE OF VERTEBRATE OXIDOSQUALENE CYCLASE
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DOI：
10.1007/bf02537826
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发表时间：
1995-03-01
期刊：
影响因子：
1.9
通讯作者：
PRESTWICH, GD
中科院分区：
文献类型：
--
作者：
ABE, I;PRESTWICH, GD
用基于机理的不可逆抑制剂[H-3]29-亚甲基-2，3-氧化橙烯对78 kDa的膜结合酶--大鼠肝脏氧化橙烯环化酶(OSC)进行了活性部位定位。该片段的氨基酸序列与酵母OSC、植物OSC和细菌角鲨烯环化酶有很高的相似性。此外，放射分析证实，高度保守区域的两个相邻天冬氨酸残基(Asp-Asp-Thr-Ala-Glu-Ala，或DDTAEA)同样被不可逆抑制剂标记。这一结果首次提供了有关OSC活性部位的结构细节的信息，并首次显示了这种脊椎动物酶与祖先真核和原核循环酶的古老谱系。有趣的是，大鼠肝脏OSC的共价修饰的DDXX(D/E)序列与其他萜类环化酶和戊烯基转移酶推测的烯丙基二磷酸结合位点序列有惊人的相似性。富含Asp的基序可能作为点电荷稳定起始的阳离子电荷。
Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, [H-3]29-methylidene-2,3-oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp-rich motif may act as a point charge to stabilize incipient cationic charge.