ON THE MECHANISM OF SQUALENE BIOGENESIS* BY H. RILLING,t T. T.- TCHEN,t AND KONRAD BLOCH CONVERSE MEMORIAL CHEMICAL LABORATORY, HARVARD UNIVERSITY, CAMBRIDGE, MASSACHUSETTS Communicated by Konrad Bloch, December 10, 1957 The discovery that the branched carbon compound mevalonic acid' (MVA) is a potent precursor of squalene and cholesterol2 has been an important advance in the understanding of polyisoprene biosynthesis. Subsequent isotopic studies have shown that the carboxyl group of MVA is lost on the way to squalene' and that the remaining 5-carbon fragment is preserved in the terpene chain without rearrange- ment of the carbon skeleton.4'5 It is therefore valid to assume that the MVA- derived condensing units are linked by interaction of C5 of one molecule with C2 of another (Fig. 1). In the present communication we wish to propose a general mechanism for the conversion of mevalonic acid to squalene. For reasons which will become apparent, squalene synthesis from branched-chain subunits will be treated as a two-stage process, namely, (a) the condensation of three "isoprenoid" units to a sesquiterpene (C15) and (b) the reductive coupling of two molecules of the C15 intermediate to squalene. The known mechanisms for the elongation of carbon chains in biological systems are condensations of the aldol-, acyloin-, or Claissen type. In all cases, at least one of the reactants has a carbonyl function and the condensation products are ketones, keto-alcohols, or glycols. On the other hand, neither C2 nor C5, the two reacting carbon atoms of MVA, has a carbonyl function, a fact which raises the question whether MVA is the precursor of a more oxidized condensing unit or whether its oxidation state remains unchanged prior to the coupling process. Earlier, we had investigated the utilization of 2-C14-5-di-T-MVA for squalene synthesis and ob- served that the C3o hydrocarbon contains the two isotopic markers in the same ratio as MVA.6 This result eliminated a condensation mechanism of the acyloin type because, on oxidation of R.CT2OH to R COOH, all the labeled hydrogen would have been removed. Moreover, it was tentatively concluded that MVA could not be utilized by way of aldehydic intermediates because the constant C14:T ratio suggested retention of both hydrogen atoms at C5 during the MVA-squalene conversion.7 Apart from the evidence to be presented below, the following ob- servations are relevant to the question of aldehydic intermediates. Shunk et al. have shown that mevaldic acid (3-hydroxy-3-methylglutaraldehydic acid) depresses the incorporation of C14-acetate into cholesterol to the same extent as MVA.8 This finding does not necessarily indicate that mevaldic acid is an obliga- tory intermediate but can be satisfactorily explained by the assumption that the aldehyde is a precursor of MVA. As reported earlier from this laboratory, dialyzed extracts of autolyzed brewer's yeast catalyze the conversion of MVA to squalene in the presence of ATP, Mn++, and pyridine nucleotides.A More recently, these extracts have yielded two fractions, A and B, which are inactive separately but will form squalene when combined.9 Fraction A, in the presence of ATP and Mn++, transforms MVA to an intermediate of unknown structure, which in turn is con- 167 Downloaded by guest on September 28, 2021 168 BIOCHEMISTRY: RILLING ET AL. PROC. N. A. S. C02H H2 HO-CHHNc 3OH H C -,C H2C OH CH2 C CH ®C "CH2OH 9 ©CH2 CH3 C CH3 ®CO2H FIG. 1.-Net change in the interaction of two molecules of mevalonic acid verted to squalene in the presence of pyridine nucleotide and fraction B. The fact that the early transformations of MVA occur in the absence of electron acceptors such as pyridine nucleotides also argues against aldehydes as obligatory intermedi- ates. It is also pertinent that attempts to demonstrate a reduction of DPN by MVA in the same enzyme system have been without success so far. The structure of the "isoprenoid" condensing units has now been further defined by experiments using a medium of 100 per cent D20 or using, as the reducing system for the MVA squalene conversion, DPN and deuterioethanol (CHSCD20H). In D20, three to four atoms of D were found to be incorporated per molecule of hydro- carbon, i.e., less than one atom per MVA or isoprenoid condensing unit. Deuterio- ethanol, in the presence of DPN, furnished less than one atom of D per mole- cule of squalene (Table 1). TABLE 1* INCORPORATION OF D INTO SQUALENE SYNTHESIZED FROM 2-C14-MEvALONIC ACID IN D2O, OR IN A MEDIUM CONTAINING 1-drETHANOL MEVALONIC ACID SQUALENEt ATOMS D PER MEDIUM (c.p.m./jsm, (c.p.m./jsM) (Atom % Excess D) MOLE OF SQUALENE D20: 1 490 43 0.11 3.6t 2 490 25 0.063 3.7 3 670 42 0.073 3.5 4 670 49 0.096 3.9 1-d2-Ethanol 670 58 0.007 0.3 * For these experiments an extract of Baker's yeast6 was centrifuged at 104,000 X g. for 2 hours. The super- natant fluid was dialyzed for 18 hours against three changes of 0.066 M (NH4)2HP04 and then lyophilized. For the D20 experiments this preparation was dissolved in twice its weight of 100 per cent D20 and lyophilized again. The particles obtained by high-speed centrifugation were washed once with 0.066 M (NH4)2HP04, combined with the lyophilized supernatant fraction, and the volume restored to that of the original extract by addition of D20 or H20. The water content of the particles is estimated to cause no more than a 2 per cent reduction of the D20 con- centration. Cofactors were added in the following concentrations: ATP and DPN, 1 mg/ml; Mn++, 0.001 M. The concentration of 2-CG-mevalonic acid was 5 X 10-4 M and of 1-d2-ethanol, when used. 0.1 M. t After dilution of the isolated squalene by non-isotopic carrier. t This sample of squalene was degraded by ozonolysis. according to Cornforth and Popjak (Biochem. J.. 58, 403, 1954). The succinic acid isolated contained 0.5 atoms of D per molecule. That the over-all transformation of MVA to squalene must be associated with the uptake of hydrogen, regardless of the mechanism involved, is evident from a con- sideration of the over-all structural changes. First, in those two molecules of MVA which become the terminal isoprene units of squalene the C C CH3 CH2COOH groups are changed to CH3 CH3 Downloaded by guest on September 28, 2021 VOL. 44Y 1958 BIOCHEMISTRY: RILLING ET AL. 169 and hence two atoms of D will be introduced into stable linkages from a D20 me- dium. Second, squalene formation from MVA, according to the equation 6C6Hl204 + 2H -> C30H5o + 6C02 + 12H20 is a reductive process, requiring a net uptake of two atoms of hydrogen. Hence from a D20 medium either one or two additional atoms of D will be taken up, depending on whether reduction occurs with H+ and H- or with 2H+ and 2e-. A minimum uptake of three or four atoms of D is therefore expected from these two processes. The same argument requires that squalene synthesis in the presence of DPN and deuterioethanol (and normal water) take place either without any uptake of D or with the uptake of one atom. From the experimental results obtained with D20 or deuterioethanol, it is clear that the events just discussed, which are independ- ent of the reaction mechanism, already account fully for the observed hydrogen incorporation. It therefore follows that none of the individual reactions by which MVA is converted to squalene can be associated with any additional uptake of pro- ton or hydride ion from the environment. When applied specifically to the coupling of "isoprenoid" units, this argument leads to the conclusion that both interacting carbon atoms, C2 and C5, retain their hydrogen throughout the condensation proc- ess. It is obvious from this argument alone that aldehydic intermediates are dis- qualified as condensing units. Another independent line of reasoning leads to the same structural restrictions for the "isoprenoid" intermediate. If the condensing units were carbonyl compounds, the product of the coupling reaction would contain oxygen. For elimination of the oxygen function, dehydration reactions leading to the formation of carbon-carbon double bonds must be invoked. The hydrogen employed for the subsequent reduc- tion of these double bonds could come from only two sources. In processes using flavoprotein as the reductant, hydride and hydrogen ions equilibrate, and hence, if double bonds were being reduced in the present case, the expected uptake of D from D20 would be two atoms for each condensing unit or a minimum of twelve for every molecule of squalene. In the event that the (hypothetical) double bonds became reduced by hydride transfer from DPND,10 the number of atoms entering from deu- terioethanol should be six. Clearly, the experimental results are not consistent with a condensation mechanism involving such unsaturated intermediates. Therefore, the polyisoprenoid chain must be established without loss and reintroduction of hydrogen at the reacting centers C2 and C5, i.e., by interaction of MVA derivatives which contain CH2 groups at both the C2 and C5 positions. As far as C5 is concerned elimination of water between C4 and C5: R*CH2CH20H R CH=CH2 will yield a methylene group that is reactive. The reactions which lead to a reac- tive methylene group at the C2 positions must be considered in conjunction with the decarboxylation step. If decarboxylation were to take place prior to condensa- tion and with formation of isopropyl derivatives, one carbon-bound hydrogen would replace each carboxyl group, introducing approximately five atoms of D into squa- lene from a D20 environment (Fig.