FUNCTION-STRUCTURE STUDIES AND IDENTIFICATION OF 3 ENZYME DOMAINS INVOLVED IN THE CATALYTIC ACTIVITY IN RAT HEPATIC SQUALENE SYNTHASE

Rat hepatic squalene synthase (RSS, EC 2.5.1.21) contains three conser ved sections, A, B, and C, that were proposed to be involved in cataly sis (McKenzie, T. L., Jiang, G., Straubhaar, J. R., Conrad, D., and Sh echter, I. (1992) J. Biol. Chem. 267, 21368-21374). Here we use the hi gh expression vector pTrxRSS and site-directed mutagenesis to determin e the specific residues in these sections that are essential for the t wo reactions catalyzed by RSS. Section C mutants F288Y, F288L, F286Y, F286W, F286L, Q293N, and Q2833 accumulate presqualene diphosphate (PSP P) from trans-farnesyl diphosphate (FPP) with reduced production of sq ualene. F288L, which retains approximately 50% first step activity, di splays only residual activity (0.2%) in the production of squalene fro m either FPP or PSPP. Substitution of either Phe(288) or Phe(286) with charged residues completely abolishes the enzyme activity. Thus, F288 W, 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 P SPP in an assay mixture complete with NADPH. P171F, Y171S, and Y171W a re all inactive. Section B, which binds the diphosphate moieties of th e allylic diphosphate subtrates, contains four negatively charged resi dues: Glu(222), Glu(226), Asp(219), and Asp(223). The two Glu residues can be replaced with neutral or with positively charged residues with out significantly affecting enzyme activity. However, replacement of e ither Asp residues with Asn eliminates all but a residual level of act ivity, and substitution with Glu abolishes all activity. These results indicate that 1) Section C, in particular Phe(288), may be involved i n 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 resi dues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a me chanism for the first reaction is proposed.