Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase
Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase
复制标题
DOI：
10.1074/jbc.273.20.12515
复制
发表时间：
1998-05-15
影响因子：
4.8
通讯作者：
Shechter, I
中科院分区：
文献类型：
--
作者：
Gu, PD;Ishii, Y;Shechter, I
大鼠肝角鲨烯合酶（RSS，EC 2.5.1.21）含有三个保守部分A、B和C，它们被认为参与催化（McKenzie，T. L.，江，G.，Straubhaar，J. R.，康拉德，D.，和Shechter，I.（1992）J.Biol.Chem.267，21368-21374）。本研究利用高效表达载体pTrxRSS和定点突变技术，确定了RSS催化的两个反应所必需的特定残基。C节突变体F288 Y、F288 L、F286 Y、F286 W、F286 L、Q293 N和Q2833从反式法呢基二磷酸（FPP）积累前角鲨烯二磷酸（PSPP），同时减少角鲨烯的产生。保留约50%第一步活性的F288 L在由FPP或PSPP生产角鲨烯中仅显示残余活性（0.2%）。用带电荷的残基取代Phe（288）或Phe（286）完全消除酶活性。因此，F288 W、F288 D、F288 R、F286 D和F286 R不能从FPP或PSPP产生角鲨烯。除Tyr（171）外，A节中的所有单残基突变体均保留了大部分RSS活性，在含有NADPH的试验混合物中未检出PSPP蓄积。P171 F、Y171 S和Y171 W都是不活跃的。部分B结合烯丙基二磷酸亚基的二磷酸部分，包含四个带负电荷的残基：Glu（222）、Glu（226）、Asp（219）和Asp（223）。这两个Glu残基可以被中性或带正电荷的残基取代，而不会显著影响酶活性。这些结果表明：1）C部分，特别是Phe（288），可能参与催化的第二步，2）A部分的Tyr（171）是催化所必需的，最可能是第一步反应所必需的，3）部分B中的两个Asp残基是活性所必需的，并且最可能通过镁盐桥结合底物。基于这些结果，提出了第一反应的机理。
Rat hepatic squalene synthase (RSS, EC 2.5.1.21) contains three conserved sections, A, B, and C, that were proposed to be involved in catalysis (McKenzie, T. L., Jiang, G., Straubhaar, J. R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374). Here we use the high expression vector pTrxRSS and site-directed mutagenesis to determine the specific residues in these sections that are essential for the two reactions catalyzed by RSS.Section C mutants F288Y, F288L, F286Y, F286W, F286L, Q293N, and Q2833 accumulate presqualene diphosphate (PSPP) from trans-farnesyl diphosphate (FPP) with reduced production of squalene. F288L, which retains approximately 50% first step activity, displays only residual activity (0.2%) in the production of squalene from either FPP or PSPP. Substitution of either Phe(288) or Phe(286) with charged residues completely abolishes the enzyme activity. Thus, F288W, F288D, F288R, F286D, and F286R cannot produce squalene from either FPP or PSPP. All single residue mutants in Section A, except Tyr(171), retain most of the RSS activity, with no detectable accumulation of PSPP in an assay mixture complete with NADPH. P171F, Y171S, and Y171W are all inactive. Section B, which binds the diphosphate moieties of the allylic diphosphate subtrates, contains four negatively charged residues: Glu(222), Glu(226), Asp(219), and Asp(223). The two Glu residues can be replaced with neutral or with positively charged residues without significantly affecting enzyme activity. However, replacement of either Asp residues with Asn eliminates all but a residual level of activity, and substitution with Glu abolishes all activity.These results indicate that 1) Section C, in particular Phe(288), may be involved in the second step of catalysis, 2) Tyr(171) of Section A is essential for catalysis, most likely for the first reaction, 3) the two Asp residues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a mechanism for the first reaction is proposed.