Gu P, Ishii Y, Spencer T A, Shechter I
Department of Biochemistry and Molecular Biology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
J Biol Chem. 1998 May 15;273(20):12515-25. doi: 10.1074/jbc.273.20.12515.
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 Q283E 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 Phe288 or Phe286 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 Tyr171, retain most of the RSS activity, with no detectable accumulation of PSPP in an assay mixture complete with NADPH. Y171F, Y171S, and Y171W are all inactive. Section B, which binds the diphosphate moieties of the allylic diphosphate subtrates, contains four negatively charged residues: Glu222, Glu226, Asp219, and Asp223. The two Glu residues can be replaced with neutral or with positively charged residues without signficantly 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 Phe288, may be involved in the second step of catalysis, 2) Tyr171 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.
大鼠肝脏鲨烯合酶（RSS，EC 2.5.1.21）包含三个保守区域，A、B和C，据推测它们参与催化作用（麦肯齐，T.L.，江，G.，施特劳哈尔，J.R.，康拉德，D.，和谢克特，I.（1992年）《生物化学杂志》267，21368 - 21374）。在此，我们使用高表达载体pTrxRSS和定点诱变来确定这些区域中对于RSS催化的两个反应至关重要的特定残基。区域C的突变体F288Y、F288L、F286Y、F286W、F286L、Q293N和Q283E从反式法尼基二磷酸（FPP）积累前鲨烯二磷酸（PSPP），同时鲨烯的产量降低。保留约50%第一步活性的F288L，在从FPP或PSPP生成鲨烯的过程中仅表现出残余活性（0.2%）。用带电荷的残基取代Phe288或Phe286会完全消除酶活性。因此，F288W、F288D、F288R、F286D和F286R不能从FPP或PSPP生成鲨烯。区域A中的所有单残基突变体，除了Tyr171，都保留了大部分RSS活性，在含有NADPH的测定混合物中未检测到PSPP的积累。Y171F、Y171S和Y171W均无活性。结合烯丙基二磷酸底物二磷酸部分的区域B包含四个带负电荷的残基：Glu222、Glu226、Asp219和Asp223。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。然而，用Asn取代任何一个Asp残基会消除除残余水平之外的所有活性，用Glu取代则会消除所有活性。这些结果表明：1）区域C，特别是Phe288可能参与催化的第二步；2）区域A的Tyr171对催化至关重要，很可能对第一个反应至关重要；3）区域B中的两个Asp残基对活性至关重要，很可能通过镁盐桥结合底物。基于这些结果，提出了第一个反应的机制。
