THE JOURNAL OF BIOLOGICAL CHEXISTI~Y Vol. 248, No. 5, Issue of March 10, pp. 1850-1867, 10i3

Printed in U.S.A.

Squalene Synthetase

SUMMARY

Studies on the properties of the reaction and the mecha- nism of conversion of farnesyl pyrophosphate to squalene have been carried out with purified yeast squalene synthetase. The general properties of the yeast enzyme are similar to those of the enzyme obtained from mammalian liver. A divalent metal ion (Mg++ or Mn++) is essential for the for- mation of presqualene pyrophosphate, but not for the con- version of this compound to squalene. However, the rate of the latter reaction is increased in the presence of Mg++. Yeast squalene synthetase activity is inhibited by concen- trations above 50 mm of farnesyl pyrophosphate, by N-ethyl- maleimide, and to a lesser extent by iodoacetamide. Hence it is evident that a sulfhydryl group or other nucleophile is required for enzyme activity.

Yeast squalene synthetase has a maximum enzyme activity at pH 7.3 to 7.5. However, great differences in enzyme activity were found, depending on the buffer used. These differences are due in part to ionic strength, and possibly to a direct effect of phosphate ion on enzymatic activity.

Initial velocity studies for the formation of squalene and presqualene pyrophosphate from farnesyl pyrophosphate have shown that the mechanism of the condensing reaction is ping-pong. Initial velocity kinetics and product inhibition studies for the reduction of presqualene pyrophosphate to squalene have shown that this reaction is sequential ordered. In the reduction reaction NADPH is the first substrate to bind to the enzyme, followed by presqualene pyrophosphate. The first product to be released is pyrophosphate, followed by squalene. The last product to leave the enzyme is NADP.

A chemical mechanism for squalene synthesis is presented.

Squalene synthctase catalyzes the contlcnaation of 2 molrcules

of farncsyl pyropliosphate and the t subscqucwt retlwtion of the

* This invcstigittion leas supported in part bv 1:escarch (;rants AM-01383 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States P\tblic IIealth Service, and G&l2417 from the National Science E’ormdation. Paper II ot this series is Reference 1.

1 On leave from the University of Chile, School of Chemistr) and Pharmacv, Santiago, Chile.

0 On leave irom the Pakistan CorlncLil of Scientific and Iudustrinl Research, I(nrxhi 39, Pakistan.

172)

intcrlrlc~tli:ltc,, prcsqu:tlci~c~ pyqhs~hntc, to squalrne (1). The

partiGpation of ncrolidyl I~~rol~llosl~ll~~tc as 811 intermediate in

this waction was suggcstcd in early work (2-4). Later Krishna.

et al. (5) rcportcd that nerolitlyl pyrophosphate is au inhibitor

of this reaction. ddditional cvitlcncc against the particil)ation

of f&c nwolidyl pyrophospllntt in squalcne synthesis was pro-

vided by Sofcr aid Rilling (6).

Krishna et al. (5, 7) reported tlw lxwcncc~ of it11 intcrmadiate of

squalenc~ synthesis ~&ich was tightly bound to enzyme. They bc-

licved this intermcdinte to be :I CaO cmhm atom compound from

its rctcntion t,ime 011 gas-liquid chromatography and its c,lution

profile on alumina column chtomato~rnphy. This intermediate

was more polar than squakwe. Lat,w Rilling (8) rcportcd the

isolation of a C&o l~~~rol)hosI)llol~~-l~ttcd intcrmcdintc of squalellc

synthesis. .\fter considcrabk wlltrovcxrs\- (4, 8-12) the chc~mi~al

structure of this coml~ountl xl:, finally wtnblished by l&~-Go-

clicmic~nl methods and by chcmicnl synthesis (13-I 5). This

coml~ound was lwncd lxwqualenc pyrophosphatc.

Tlic over-all wactioii catalJ.zcd by squalci~c syrithetasc occurs

in two consecutive st,cps (Fig. I). In the first, 2 moledw of

farnwyl pyrophosphatc, are condctnscd to form prcsqualcnc pyre-

pllospllate (the CSO intcrnwdiatc) with tlie rclcasc of a pyrol)lios-

pliatc moiety. In the second reaction, lnwqualrne pyrophos-

phate is reduced by N;\I)l’II to squak~~w.

Two diffcrcnt concwtcd mrchanisrns have been propowd for

the: first partial rwction of sql&lw synthesis. The mcchnnisms

l~rolxxwl by Rilling el nl. (I 3) alld by I~Xmond cl al. (12) involve

ail atta(sk of the 7r c,lcctrolls of carbon 2 of OI~C Inrnrsyl pyrophos-

phte on carbon 1 of tlic othrr. This attack is hc~lpetl by a iiw

ck~olhilic group m the cwzyme. ‘l’lic formation of the cycle-

propane ring thc~~ results from a conwrtcd attack on the carbon

atom bound to the cnzymc with the release of a lwoton from C-l

of the incoming farncsyl l~yrophosplintc molcculc.

Iii the n~cclini~ism ~nx~po.~ed by va11 Tamclcii and Schwartz

(16) the r system of carbon 2 of one farncsyl pyrophosphatc

nttwks carbon 1 of the othw fnrnesyl ~)yropIiosphatc with cithcl

intrarnolccular migration or extwlal addition of pyrophosphate.

An isomerization an:do~ous to that, cxtalyzcd by isopentenyl

pyrophoaphatc isomcraac (17, 18) 1lwn I’ollows. AIL attack of the

newly formed double bond on the carbon atom binding l)yro-

phosphate, with sulxwqucnt elimina.tion of p)-rophosphatc, would

yield the cycloproI)atlc~ ring. Collalw of the rarbonium ion 1~)

elimination of a proton from tllc tertiary carboll, would then re-

sult in the foimation of presqualellc 1)?-roI)11osl)liate. l<oth of

tlw above mccllanisnis rwult in tllc elimination of one of the

hydrogen atoms from wrbon 1 of ow of the two condcllsing far-

1856

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O-P-O-P

P-o-P-0

:- PPi

O-P- O-P

/--- NADPH f H+

\, I NADP’S PPi

FIG. 1. Reaction sequence in the enzymatic conversion of farnesyl pyrophosphate to squalene.

nesyl pyrophosphate moleculca as ~vas demonstrated by PopjBk (19).

The reduction of prcsqualene pyrophosphate to squalene is be- lieved to involve as a first step the elimination of pyrophosphate (13, 14, 16). The carbonium ion formed would then undergo ring cspansion to yield a cyclobutonium ion. The cyclobuton- ium ion would bc opened and stabilized by the presence of allylic double bonds. Hydride transfer from NADPH would finally yield squalrne as the product.

X second proposed mechanism (12) consists of a two-step reac- tion. In the first step, rtarraiigement of the presqualene pyro- phosphate molecule results in ring expansion and migration of the pyrophosphate moiety. In the second step an attack by the hydride ion from NAIDl’H ~vould result in ring opening and the elimination of the pyrophosphatc anion.

In the present study we have attempted t.o work out the mecha- nism of squalcnc synthesis through kinetic analysis. Initial velocity kinetics of this rcactioii show that the first partial reac- tion is ping-pang. The second reaction, the reduction of prc- squalene pyrophosphate, is however sequential and ordered. From t,his information we have proposed a new mechanism for the synthesis of squalene.

EXPERII\fESTAL PROCEDURE

dlalerials-The materials used in this study wcrc purchased from the following sources: Tween 80 from E. I-I. Sargent; l- butanol, benzcnc, and ethyl acetate from Fisher Scientific Lab- oratories; Silica Gel H and Silica Gel G from E. Merck; NhDPH from Schwarz-hIann Laboratories; Good’s buffers from Cal- biochem; and Tris base from Sigma Chemical Co. Xl1 other re- agents were of analytical grade.

The preparation of squalcnc synthetase and [‘“C]- and [I%]- fmncsyl pyrophosphatel were reported previously (I, 20). In

1 The abbreviations used are : [*“Clfnrnesyl pyrophosphnte, [4, 8, 12-14C]fsrncsyl pyrophosphate; [‘%]presqualene pyrophosphate, [3’,7’, II’, 1”,5~,9”-14C]prcsqllalrle pprophosphate.

each of the experiments reported in this paper the enzyme ob- tained after DE-1E-cellulose column chromatography was used. Assays for the ov~all conversion of farnesyl pyrophosphate to squalene and the first partial reaction; i.e. the biosynthesis of prcsqualenr pyrol)hosphate, were also reported previously (1, 20). Concentrations of the substrates used are shown in the legends to the figures.

Preparation of Presqualene Pyrophosphate-Two diffrrent prep- arations of presqualene pyrophosphate were used. Low specific activity prcsqualcne pyrophosphate [3’, 7’, I I’, I”, 5”, 9”-14C]- (1,2-ar~ti-l,3-ar~ti)-3-(2’,6’,lO’-trir~~cthylur~dcca-l’,5’,9’-aZZ frans- tricnyl)-2-(4”, 8”-dimethylnona-3 “, 7”Wl frans-dicnyl) 2-methyl-l~mcthyl 1)yrophosphate cyclopropane was synthesized enzymatically in the following incubation mixture: potassium phosphate buffer, $1 7.4, 100 xnnr; magnesium chloride, 10 rnM; Tween 80, 2 mg; [Wlfarnesyl pyrophosphate (4 x lo6 dpm), 1.37 rnAf; and DEhE-cellulose-purified yeast squalenc synthetase, 16.5 mg of protein; in a total volu~rlc of 1 liter. After 4 hours of incubation under nitrogen, the reaction was stopped by the addi- tion of 150 ml of concentrated ammonia and 150 ml of 0.25 M EDTA. The solution was incubated for an additional 10 min at 25”. The [l”C]lncsqualenc pyrophosphate formed was extracted with 500.ml portions of I-butanol until no radioactivity was found in the 1 -butanol phase (generally six extractions). The I-butanol was removed under vacuum by Hash evaporation. The pale yellowish syrup was dissolved iu benzene and the solvent was again evaporated. This process was repeated two more times to rcmovc all traces of 1 -butanol. The final syrup was dissolved in a minimum amount of benzene and applied to a se- ries of glass plates, 20 x 20 cm, coated with Silica Gel H (0.5 mm thick). The thiii layer plates were developed with ethyl acetate-isol)l.ol)vl alcohol-ammonia (45 : 60 :45) ; (RF values were the following: presqualene pyrophosphatc, 0.40; prcsqualenc monophosphate, 0.75; farnesyl pyrophosphate, 0.20; and far- nesyl phosphate, 0.70). [WJPrcsqualene pyrophosphate was located by assaying l-cm strips of silica gel scraped from the plate for radioactivity with a Tri-Csrb liquid scintillation spcc- trometcr. The remainder of the desired region of the plate was scraped into a bcakcr and the [14C’]presqualene pyrophosphate was eluted with 1 yc ammonia in methanol. Rcchromatography of this material on thiii layer plates (Silica Gel II) with isopro- pyl alcohol-roncclltl,ated arllrllonia-\~-ater (6 : 3 : 1) (4) showed a single spot of ra(lioactivity (RF 0.40). Reverse phase thin layer chrom:ltogl~:rl~l~~ (20) of the acid hydrolysis products gave a single zone of radioactivity (R, 0.65), coincident with carrier acid-treated. presqualene alcohol. Mass spectra of the purified [14C]~~rcsq~~:~Ic1~e pyrophosphate agreed with that ob- tained by Edmond et al. (12).

High specific activity [‘%]presqualene pyrophosphatc, labclcd in the same positions as the material of low specific activity, was preparcd uildcr conditions similar to those reported above. The following components: potassium phosphate buffer, $1 7.5, 50 m&r; magnesium chloride, 10 mM; Tween 80, 200 g; [i4C]farnesyl pyrophosphate (2 x lo7 dpm), 9.61 nmoles; and purified squalcne synthetase, 2 mg of protein were incubated, in a total volume of 10 ml, at 25” for 90 min. The reaction was stopped by the addi- tion of 20 ml of 957, ethanol, and the reaction mixture was ex- tracted three times with 5 ml of petroleum ether. Concentrated ammoilia, 1.5 ml; EDT& 0.25 M, 3 ml; and water, 50 ml, were added and the solution was incubated for 15 min at 25”. The incubation mixture was extracted with three 100.ml aliquots of l- butanol, and thcii with three lOO~m1 aliquots of benzene. The l-butanol and bcnzcne extracts were combined and the solvent

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was removed under vacuum by flash evaporation. The residue was extracted first with l-butanol-benzene (1: I), and then with 2% ammonia in methanol. The extracts were combined, the solvent removed by flash evaporation, and the residue applied to a series of thin layer plates of Silica Gel H. The plates were developed with ethyl acetate-isopropyl alcohol-ammonia (45 : 60 : 45). The [Wlpresqualene pyrophosphate region was scraped from the plate and eluted with 2% ammonia in methanol. Thin layer chromatography of the eluted material showed a single zone of radioactivity on chromatography on Silica Gel H in a system of isopropyl alcohol-ammonia-water (6 :3 : 1). A single compo- nent was also observed on reverse phase chromatography of the acid hydrolysis products.

Assay for Reduction of Presqualene Pyrophosphate-The rat,e of reduction of presqualene pyrophosphate was determined in an incubation mixture that contained potassium phosphate buffer, pH 7.4, 25 pmoles; magnesium chloride, 5 pmoles; [14C]presqua- lene pyrophosphate, 24 nmoles and 20,000 dpm; NADPH, 0.8 pmole; glycerol, 150 ~1; and purified squalene synthetase, 5 pg of protein, in a total volume of 0.5 ml. Aft’er 2 to 10 min of in- cubation at 37” the reaction was stopped by the addition of 1 ml of 95% ethanol. The squalene formed was extracted with three 2-ml portions of petroleum ether. Carrier squalene, 0.5 mg, was added and the petroleum ether extract was evaporated to dryness under a stream of nitrogen. The residue was dissolved in petro- lcum ether and spot,ted on a thin layer plate of Silica Gel G, 0.3- mm thickness. The chromatogram was developed with petro- leum ether-ethyl ether (98 : 2). After development, the plate was air-dried and sprayed with 1 y0 iodine in methanol. The squalene zone was scraped from the plate into a glass vial and radioactivity was measured by assaying in a Tri-Carb liquid scintillation spec- trometer as described earlier (21).

Phosphate Determinafion-The concentration of substrate was determined by measuring the concentration of phosphate by the method of Galliard et al. (22).

Protein Determination-Protein was determined by the method of Gornall et al. (23).

Data Processing-Reciprocal velocit,ies were plotted against the reciprocal of substrate concentration. Similar plots were made earlier by Dugan and Porter (24) for t,he over-all reaction catalyzed by pig liver squalene synthetasc. The first partial reaction of squalcnc synthesis follows the general equation for a ping-pong mechanism (25) where

VA” v= (Ka+Ka,)A+A” (1)

and K, and K,! are the Michaelis constants for the 1st and 2nd molecules of farnesyl pyrophosphate that bind to the enzyme; B is the farnesyl pyrophosphate concentration; 1’ is the maximum velocity, and v the rate of the reaction.

The data obtained from the initial velocities for the second partial reaction, the reduction of presqualene pyrophosphate to squalene, fit Equation 2. This equation fits a sequential Or- dered Bi Ter reaction where

VBC y = KcKib + K,,C f KcB + BC (2)

and B and C represent the concentrations of NADPH and prc- squalene pyrophosphatc, respectively; & and li, the Michaelis constants for the respective substrates, and Iii5 the dissociation constant for NADPH.

For product inhibition studies Equation 3, or parts of it, were used :

v1v2 - PQR

V,V,BC - Ke

v = V,KcKib t V,KcB i V,K,,C + V,BC f V,K KirBP +

K K. eq lb

V,KirK qP+

V,K. K V,K V,K

K K1qp Re2PQ++PR +

K -4 eq =l eq

v’“p VI V,R ‘J,KirKq

K QR c x PQR i 1 BPQ +

K K. eq lb K K. K.

BCP +

-3 -l eq lb xc

V,K; K p I V, Kb VIKr

K K. K. BcQ L K. CQR +

K K. K. BCPQ i

eq lb xc 1q lr eq 1c lb

IV.? Kb

K. K. K. CPQR

1q Ip 11‘ (3)

In this equation P, Q, and R are the products of the reaction; namely, pyrophosphate, squalenc, and NADP, respectively. The other symbols follow Cleland’s nomenclature (25, 26). The data were fitted to the above equat’ious, assuming equal variance for the velocities (27).

RESULTS

General Properties oj” Reaction

ICJect OJ Protein Concentralion and Time of Incubation-En- zyme activity for the over-all conversion of farnesyl pyrophos- phate to squalene and for the formation of presqualene pyrophos- phate (Fig. 2,9 and B) was proportional to the amount’ of proteiu up to 50 to 60 pg per ml. In the second partial reaction, the re- duction of prcsqualenc pyrophosphate to squalene, activity was linear with protein added to at least 30 pg per ml (Fig. 2C).

Enzyme activity for each of the above reactions was also linear with time, up to approximately 30 min (Fig. 3, A, B, and C).

pH Optimum and IZffect of Tonic Strength-Yeast squalene syn- thetase showed maximum enzyme act,ivity at pH 7.3 to 7.5 (Fig. 4). In addition enzyme activity was greater in phosphate buffer than in the buffers described by Good (28) and in Tris-HCl. To test whet’her activation by phosphate ions is a specific or nouspe- cific ionic strengt’h effect, experiments were performed with 50 mM Buffer A (N-tris(l~ydror~metl~~l)n~ethyl-2~aminoethane sul- fonate), pH 7.5, and different concentrations of potassium phos- phate or KU. The result,s of thcsc cspcriments (Fig. 5) show that the effect of phosphate ion is not completely specific. KC1 replaces phosphate ions at high osmolarities. However, a specific effect of potassium phosphate cannot be ruled out since it was a better activator at low osmolarities. An increased enzyme ac- tivity iu higher ionic strength buffer was observed for each of the three reactions studied (Fig. 5).

Metal ion-Squalene synthrtase requires a divalent metal ion for enzyme activity. Magnesium ion is essential for the over-all reaction (Fig. 6A) and for t,lle formation of presqualene pyro- phosphate (Fig. BB). When 30 ~molcs of EDTA were added instead of magnesium (the zero magucsium concentration, Fig. GA), no reaction was observed. Magnesium ion is not essent’ial for the reduction of presqualene pyrophosphatc to squalcne, but the reaction rate is enhanced nearly 50 times in the presence of 30 rnY magnesium chloride (Fig. BC).

Manganous ion can replace magnesium ion as an act,ivator for the over-all reaction (Fig. 7). Masimum activity was reached at a concentration of approximately 1 mnl. At higher conccn- trations manganous ion was inhibitory.

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0:. - L7 -.--.& .I2

PROTEIN IN mg

FIG. 2. The effect of the concentration of squalene synthetase tained the same components as reported for Fig. 2A, except for on the rate of synthesis of presqunlene pyrophosphate and squa- the omission of NADPH. The assay was carried out as described lene. A, the effect of protein concentration on the conversion of previously (I, 20). 6, dependence of the rate of reduction of farnesyl pyrophosphate to squalene. The following 1%.ere incu- presqualene pyrophosphate to squalene on protein concentration. bated in a total volume of 1 ml at 37” for 5 min: potassium phos- The follo~ving were incubated in a total volume of 0.5 ml for 5 min phate, pH 7.5, 50 mM; MgC12, 10 mu; NADPH, 1.6 mM; glycerol, at 37”: potassium phosphate buffer, pH 7.4,50 mM; MgC12, 10 maI; 0.3 ml; and [l%]farnesyl pyrophosphate, 0.05 rnM and 20,000 dpm. The amount of prot,ein used in the incubation mixture is given on

[Wlpresqualene pyrophosphate, 0.048 mM and 20,000 dpm; NADPH, 1.6 mM; and glycerol, 0.15 ml.

the figure. The conditions of assay were reported previously (1, The amount of protein

used in each assay is shown on the figure. The conditions of assay 20). 13, dependence of the rate of presqualene pyrophosphate are reported under “Experimental Procedure.” formation on protein concentration. The incnbationmfxture con-

b” REACTION T:k IN MINUTES

E’Lc. 3. Dependence of synthesis of presqualenc pyrophosphate and squalene on time. A, the effect of time on the conversion of farnesyl pyrophosphate to squalene. The following mere incu- bated in a total volume of 1 ml at 37” for variable periods of time: potassium phosphate buffer, pH 7.5, 50 mM; MgC12, 10 mM; NADPH, 1.6 mM; glycerol, 0.3 ml; [‘“Clfarnesyl pyrophosphate, 0.05 mM and 20,000 dpm; and purified squalene synthetase, 40 pg of protein. The conditions of assay were reported previously (1, 20). B, the effect of time on t,he synthesis of presqualene pyro- phosphate. The incubation mixture was the same as reported in

Fig. 3A, except for the omission of NADPH. Assays for enzyme activity were carried out as described previously (1, 20). C, the effect of time on the reduct’ion of presqualene pyrophosphate to squalene. The following were incubated in a total volume of 0.5 ml for variable periods of time at 37”: potassium phosphate buffer, pH 7.4, 50 mM; MgCL, 10 mM; [‘*Clpresqualene pgrophosphate, 0.048 mu and 20,000 dpm; NADPH, 1.6 mM; glycerol, 0.15 ml; and purified squalene synthetase, 5 fig of protein. The conditions of assay are reported under “Experimental Procedure.”

IrLhibitors-Fluoride ion has been used by ot,her investigators,

and by us, to inhibit a very active phosphatase present in the microsomes and in crude preparations of squalene synthetasc. Fig. 8 shows the effect of fluoride ion on purified bakers’ yeast squalene synthetase. Fluoride ion is a potent iuhibitor of the three reactions studied. Fifty per cent inhibition was observed at fluoride concentrations brtlvecn <5 to 8 mM.

Krishna et al. (5) reported that pig liver squalene synthet’ase is strongly inhibited by p-h-drosvlnrrcuriberlzoate and N-ethyl-

maleimide. We obtained similar results with the purified bakers yeast squalene synthetase. The inhibition of squalene synthe- tase by reagents that combine with sulfhydryl groups is shown in Fig. 9, A, R, and C. The condensat,ion of farnesyl pyrophosphate t,o form presqualene pyrophosphate and the reduction of the latter to squalene are inhibited to the same extent, indicating that each of the partial reactions of squalenc synthesis requires either a sulfhydryl or a nucleophilic group. Iodoacetamide, another reagent that combines with sulfhydryl groups, was not as effec-

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PH

f li 9 8

FIG. 4. The effect of pH on the rate of squslene synthesis. The assay mixtrrre contained: MgCls, 10 rnv; glycerol, 0.3 ml; [14C]famesyl pyrophosphate, 0.05 mM and 20,000 dpm; and purified squalene synthetase, 40 pg of protein, in a total volrlme of 1 ml. The incubation time was 5 min. Synalcne \\-as extracted and purified as reported previously (1,20). The following buffers were used at a concentration of 50 mM: O---O, Tris-HCl; O--O, potassium phosphate; and n--A, Good’s buffers (pH 5.55, 6.31, and 6.60); 2-(A-morpholino)ethane sulfonate (pH 6.70, 7.10, and 7.31) ; BnfYer A (pH 7.75) ; and Tricine, pH 8.50.

tive an inhibitor of squalene synthesis as LVetllylmaleimide. This result was also observed with rat liver squalene synthetase

(29) Farnesyl pyrophosphate at high conccutrations is an inhibitor

of yeast squalcne synthetase. Xtasimal activity was obtained with a farnesyl pyrophosphate concentration of 25 PM. At 600 PM, the enzymatic activity was 32% of the mnsimal activity (Fig. 10).

Reduced Nucleotide Specificity of Xqunlene Sunthetase-l’revi- ouslg Shechter and I{loch (30) analyzed yeast squalene synthe- tase for its requiremeut for pyridine nucleotidcs. ;Z similar anal- ysis of our enzyme preparation for pyridinc, nuclcotide specificity is shown in Table I. NADH can replace NADI’H; however, the reaction proceeds at a much lower rate.

Formation OJ" Lycopersene-Yeast squuleuc syuthctase con- denses 2 molecules of gernuylgeranyl pyrophosphate to form the Cqg compound lycopersene, Table I. Howel-cr, the rate of this reaction is much slower than that for the formation of squalene.

Initial Velocity Kinetics

Over-all Reaction-A series of kinetic espcriments was carried out to obtain data on the mechanism of a&ion of yeast squalene synthetase. When NADPH was the variable substrate, and four fixed farnesyl pyrophosphate conccnt,rations wrre used, a parallel patteru of initial velocities was obtaiued for the reciprocal plots (Fig. 11). From replots of the intercepts of the above lines the Michaelis constants were determined for farnesyl pyrophosphate

(4.4 x 1O-7 M) and for NhDPI-I (6.1 X IO-” RI). The parallel pattern of initial velocities obtained (Fig. II), iudicat.es that the additions of farnesyl pyrophosphate and N-iDPI- to the enzyme are irrcvcrsibly connected. When the reciprocal of the coiicen-

0.1 02 0:

IONIC STRENGTH IN OSMOLARITY

FIG. 5. l<Xect of ionic strength on the rates of the reactions cat,alyzcd by squalene synt,hetase. The conditions of assay were Buffer A, pH 7.5, 50 mM; MgC12, 10 mM; NADPH, 1.6 mM; nnd glycerol, 30Llc. Purified sqllalene synthetase, 10 fig of protein, was Ilsed in the over-all conversion of farncsyl pyrophosphate to sqnxlene and in the formation of presqualene pyrophosphate. In the reduct,ion of prcsqualene pyrophosphate to squalene 5 pg of protein were used. The concentration of [‘%]farnesyl pyro- phosphate was 0.05 rnsx and 20,000 dpm for the over-all reaction and for the formation of presqualene pyrophosphate. The [14C]- prcsqualene pyrophosphate concentration w-as 0.048 rnM and 20,000 dpm in the reduction of presqualenc pyrophosphate to sqlxalene. The react,ion for t,he formation of presqualene pyrophosphate was carried out in t,he absence of NADPH. O-0, t,he effect, of phosphat,e ion on the over-all conversion of farnesyl pyrophos- phate to squalene; A--A, the effect of KC1 on the formation of presqualene pyrophosphate; O---O, the effect of KC1 on the re- duction of presqllalene pyrophosphate to squnlene; and O--O, the cffcct of KC1 on t,he over-all reaction.

tration of farnesyl pyrophosphatc was ljlottcd against the recail)ro- cal of the rate of reaction, \vith NADPH held at several fiscd sub- strate concentrations, a family of parallel straight lines was obtained, thus ilidicating that tllc binding of the 2 molecules of farnesyl pyrophosphat,e are irrcvcrsibly connertcd. H~nc(,, a ping-pang-l;ypc: mechanism is deduced for the first partial rcnction of squalene synthesis.

First Parlial Reaction-To further t,rst this possibility, initial velocity studies of the first partial reaction were conducted. If the mechanism for the formation of presqualenc pyrophosphate is ping-pang, the reaction raLtc would follow Equation 4.

1 Ka + Ka, -= v V $ ++

This equation lmxdicts a straight line when a reciprocal plot of the data is made. If the reaction is sequential, the data would follow Equation 5, which predicts a parabolic line upon plot,ting the reciprocals.

1 K. K ma’ 1

-=-za+ Ka + K.3,’ 1 (5) v V v -p+

When the initial velocity csprrimcnt for the first partial reaction was carried out (Fig. 12)) a straight line was obtained. A value of 3.9 x 1O-7 was obtained for (K, + K,!), from the estrapola-

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lS61

I 0 0

IO 20 30

Mg Cl2 hM) IO 20 30

MgCkhM)

Fro. 6. Effect of MgCls concentration on the rates of the reac- tions catalyzed by squalene synthetase. A, dependence of the rate of the over-all conversion of farnesyl pyrophosphate to squdlene on the concentration of magnesium ion. The following were incu- bated in a total volume of 1 ml for 5 min at 37”: potassium phos- phate buffer, pH 7.5, 50 rnM; NADPH, 1.6 mix; [l%]farnesyl pyro- phosphate, 0.05 rnM and 20,000 dpm; glycerol, 0.3 ml; and purified squalene synthetase, 10 .~g of protein. The concentrations of MgClz are reported on the figure. The assay for enzyme activity was carried out as reported previously (1, 20). B, dependence of rate of synthesis of presqualene pyrophosphate on the concentra-

;i

cl

5 IO

MnCIz (mM)

FIG. 7. Effect of concentration of manganous ion on the rate of synthesis of squalene from farnesyl pyrophosphate. The assay mixture was the same as reported in Fig. BA, except for the replace- ment of MgClz by MnClr.

tion of the line. This value is in good agreement with the Mich- aelis constant for farnesyl pyrophosphate calculated from the re- sults of initial velocity studies of the over-all reaction.

With this set of experiments we were able to define the order of

o-

O-

3

IO 20 30 MgClz (mM)

tion of MgC12. The incubation mixture was the same as in Fig 6A, except for the omission of NA4DPH. The assay for enzyme activity was carried out as described previously (1, 20). C, de- pendence of the rate of redllction of presqllalene pyrophosphatc to sqllalene on the concentration of MgCls. The following were in- cubated in a total volume of 0.5 ml at 37” for 5 min: potassium phosphate buffer, pH 7.4, 50 mM; [14C]presqualene pyrophosphate, 0.048 rnM and 20,000 dpm; NAIIPH, 1.6 mM; glycerol, 0.15 ml; and purified squalene synthetase, 5 pg of protein. The conditions of the assay are reported under “EZxperimental Procedure.”

addition and release of subst,rat,es and products for the first partial reaction.

Second Partial Reaction-The nest series of experiment,s was designed to study the second partial reaction. Initial velocity studies (Fig. 13) showed an intersecting pattern when the rc- ciprocal of NADI’H concentration was plotted agLtinst the re- ciprocal of the rate of the reaction. From replots of the intercept against the reciprocal of the concentration of the changing fixed substrate, the Michaelis constants for NADl’H and presqunlene pyrophosphate, were calculated. Values of 7.0 x 10e5 and 7.7 X lop7 M were obtained for KADI’I-I and prcsqualcne pyrophos-

phate, respectively. The intersecting pattern obtained for the reduction of pre-

squalenc pgrophosphate indicates that the second partial reaction is sequential, i.e. that both substrates must be bound to the En- zyme before the release of product.

Product Inhibition Studies--In o&r to determine whet&r this reaction is ordered or random, and also to know the order, if any, of the addition of substrate and rclrase of products, experiments were carried out on the inliibit,ion of t,he enzyme reaction by its products. The effect of inorganic pyrophosphate on the second partial reaction catalyzed by squalene synthetase is shown in Fig. 14. When the reciprocal of the rcxaction rate was plotted against the reciprocal of the concentration of NADPH, at a constant nonsaturating concentration of presqualene pyrophosphntc, an S- linear, I-linear noncompetitive inhibition was observed. When NADP was used as product inhibitor, and presqualene pyrophos- phate the variable substrate, an S-linear, I-linear noncompetitive inhibition was obtained (Fig. 15). When NADPI-I was the variable substrate and NADP was the inhibitor, double reciprocal plots showed a pattern of S-linear competitive inhibition (Fig. 16).

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KF (mM)

Fro. 8. Inhibition of squalene synthetase by potassium fluoride. The effect of potassium fluoride on the over-all conversion of farnesyl pyrophosphate to sqllalene (0). The incltbation mixture contained: potassium phosphate buffer, pH 7.5, 50 mM; MgClz, 10 mu; NADPII, 1.6 mM; glycerol, 0.3 ml; [‘%]farnesyl pyrophos- phate, 0.05 mM and 20,000 dpm; and enzyme protein, 20 pg. The total volume was 1 ml and incubations xl-vere carried out for 5 min at 37”. The concentration of potassium fluoride llsed is shown on the figure. Inhibition of the synthesis of prcsqualene pyrophos- phate by potassium fluoride (A). The incltbation mixture was the same as for q except for omission of NA1)PI-I. Assays were carried ollt as described previollsly (1, 20). Inhibition of the re- duction of presqualene pyrophosphate to sqLlalene by potassium fluoride (0). The following n-ere incubated in a total volume of 0.5 ml for 5 min at 37”; potassium phosphate buffer, pH 7.4, 50 maI; MyCla, 10 mnl; [14C]presqunlenr pyrophosphate, 0.048 mM and 20,030 dpm; NADPH, 1.6 mu; glycerol, 0.15 ml; and prlrified sqlla- lene synthetase, 5 fig of protein. The conditions of assay are re- ported lmder “12xperimenlal Procedure.”

Purified squalene synthrtase catalyzes an over-all reaction in which three substrates and four products are involved. The over-all rcnction is in turn composed of t’wo partial reactions, namely, the condensation of 2 molrcules of farnesyl pyrophos- phate to form presqualene p~rol)ho.~phatc, and the reduction of this intermediate to form squnltnc.

The reaction for the formation of squalene is linear with respect to the conccnt,ration of protein, up to 60 pg p’r ml and with time, to 30 min. A marked deviation from linearity was obscrvcd, however, when more than 45;; of the fnrnesyl pyrophosphate was used.

Bakers’ yeast squalene synthetusc has an optimum pH of 7.3 to 7.5. The same optimum pH was found for the rat (29) and pig liver (5) enzymes. Yeast squalene synthetnse showed large diffcrcnces in activity in the presence of different buffers even though thcg were used at identical molar concentrations. The opt,imum ~1-1 was, however, the same for all the buffers tested.

The increased activity of squalene synthetasc observed with phosphate ion could be due to (n) n specific effect of this ion, (b) a nonspecific effect of ionic strength, or (c) to both effects. The results reported show that at high osmolaritics, KC1 can replace phosphate ion. However, the phosphate ion is a better activator

for squalene synthesis at low ionic strength, thereby indicating that bot,h high ionic strength and phosphate ion are important for maximum activity of squalene synthetase. Ionic strength and phosphate ion are also important for both partial reactions of squalcnc synthesis.

A divalent metal ion is an absolute requirement for activity of yeast squalene synthetane. Either magnesium or manganese xvi11 meet this requirement. At low concentrations rnaJlganous ion is a better activator than magnesium ion, but at higher concentra- tions manganous ion is inhibitory. This phenomenon has been obaervcd with other enzymes as well, eapccially those which have phosphorylatctl intermediates (31-33). Activation of squalcne synthctasc by magnesium ion showed a biphasic curve for the three reactions studied; first, activity rapidly incrcascd with an increxsc in magnesium ion concentration, up to 2.5 to 5 mM, and then it incrcascd more slowly up to 30 n1M (the highest conccn- tration tested). This secondary incrcasc in cnzymc activity at high conccJnbrations of metal ion could be due to an increase in ionic strength in the incubation mixture (compare with Fig. 4). Magnesium is an absolute requirement for enzyme activity for the first part.ial reaction, but it does not appear to be essential for the reduction of presqualene pyrophosphate. The second partial reaction occurs at a significant rate, even in the presence of 30 111~ EDT-1 and zero magnesium concentration (Fig. 6). However enzyme activity for this partial reaction is increased greatly in the presence of magnesium ion.

Fluoride ion has bern widely used to inhibit a microsomal phospllatase in crude preparations of squalene synthetase (5, 6, 10, 31). We have found, however, that lluorida ion is a potent inhibitor of squalrne syuthetase and its inhibitory action has been dcmouatratcd for b&h part,ial reactions of squalene synthe- SlR.

The diffcrc,ncc in inhibition of squalcne synthctasc obtained with ids-etll!-lrnaleiinide and monoiodoacctamide cannot be es- plained at prrscnt. Ilowever, the same phenomenon has been observed by othrrs (29, 34, 35). It appears very probable that squalene synthctase requires a sulfhydryl group for full activity. This fact is supported not only by the inhibition studies but also by the rcquircmcnt of 2-mercaptoethanol or dithiothreitol for stabilizat,ion of the enzyme. Krishna et al. (5) and Goodman et al. (29) previously reported inhibition of the over-all reaction of squalcnc synthesis by reagents that combine with sulfhydryl groups WEWJL pig and rat liver squalene synthetases were used.

Both inhibitors, ,V-ethylmalcimide and monoiodoacetamidr, inhibited squalcnc synthetasc to nearly the same extent in each partial reaction (Fig. 9), thus indicating that a sulfhgdryl or a nuclcophilic group is important in each reaction.

Krishna et al. (5) found that high concentrations of farnesyl yyrophosphate are inhibitory for pig liver squalcnc synthetase. Our experiments with yeast squalenc synthetase also showed that this enzyme is inhibited by farnesyl pyrophosphate. However, the inhibitory concentrations of farnesyl pyrophosphate that we found were higher than those required for the critical micellar concentration for farnesyl pyrophosphate. Possibly the inhibi- tion by farnesyl pyrophosphate that we observed could be due to a nonspecific detergent effect. Shechter and l{loch (30) also re- ported inhibition of squalelw synthetase by other detergents such as dcosycliolate.

Dugan and Porter (24) reached maximum velocity for squalene synthesis at 0.2 to 0.4 PM farnesyl pyrophosphatc. They es- plained their results on the basis of micelle formation. We did not observe maximum velocity at this farnesyl pyrophosphate concentration with our assay procedure. Possibly this differ-

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1563

P ,o’

I

IO-

o-

FIG. 9. Inhibition of squalene synthetase by monoiodoaceta- mide (0) and N-ethylmaleimide (0). Incubations were carried out in the presence of 2-mercaptoethanol-free squalene synthetase and the inhibitor at the concentrations indicated on the figure for 10 min at 37”. An aliquot of this solution was then transferred to the reaction mixture to start the reaction. A, inhibition of the over-all conversion of farnesyl pyrophosphate to squalene. The incubation mixture contained: potassium phosphate buffer, pH 7.5, 50 mM; MgC12, 10 mM; NADPH, 1.6 mM; glycerol, 0.3 ml; [%I- farnesyl pyrophosphate, 0.05 mM and 20,000 dpm; and purified squalene synthetase, 10 pg of protein. Incubations were carried out for 5 min at 37”. The conditions of assay for squalene syn- thesis were reported previously (1, 20). B, inhibition of the

FARNESYL PYROPHOSPHATE (mM)

FIG. 10. The effect of concentration of farnesyl pyrophosphate on the rate of synthesis of squalene. The incubation medium con- tained: potassium phosphate buffer, pH 7.5,50 mM; MgC12, 10 mM; NADPH, 1.6 mM; glycerol, 0.3 ml; purified squalene synthetase, 1Opg of protein; and farnesyl pyrophosphate in the concentrations indicated, in a total volume of I ml. Incubations were carried out at 37” for 1, 2, and 5 min. The conditions of assay for squalene n-ere reported previously (1, 20).

ence in results is due to the presence of glycerol in our system

which increases the solubility of farnesyl pyrophosphate in the

incubation mixture.

Mechanism of Reaction-The results of initial velocity expcri-

merits for the over-all react.ion of squalene synthesis indicate

synthesis of presqnalene pyrophosphate by iodoacetamide and N- ethylmaleimide. The incubnt,ion mixture u-as the same as re- ported in Fig. 9A, except for the omission of NADPH. The assay for presqualene pyrophosphate was carried out as reported previ- ously (1, 20). C, inhibition of the reduct,ion of presqualene pvro- phosphate to squalene by iodoacetamide and N-ethylmaleikide. The following were incubated in a total volume of 0.5 ml for 5 min at 37”: potassium phosphate buffer, pH 7.4, 50 mM; MgC12, 10 IIIM;

[‘%]presqualene pyrophosphate, 0.048 m&f and 20,000 dpm; NADPH, 1.6 mM; glycerol, 0.15 ml; and purified squalene synthe- tase, 5 pg of protein. The conditions of assay are reported under “Experimental Procedllre.‘!

TABLE I

Activity of squalene synthetase with substrate analogs

The assay mixture contained potassium phosphate buffer, pH

7.0, 50 mnl; reduced pyridine nucleotide, 1.6 rnM; glycerol, 0.3 ml; purified squalene synthetase, 5 pg of protein: and either [4,8,12- ‘%]farnesyl pyrophosphate, 0.05 ml\1 and 20,000 dpm, or [4,8,12,

16-Wlgeranylgeranyl pyrophosphate, 1 JAM and 24,400 dpm. The assay procedures for sqnalene and lycopersene were reported previously (1, 20).

Substrate IIYdrocroc~~ons

v/c

NADPH, [14C]farnesyl pyrophosphate. 100.04 NADPH, [l%!]geranylgeranyl pyrophosphate. 6.3” NADH, [Wlfarnesyl pyrophosphate.. 37.4a

a Squalene. 6 The product isolated was identified as lycopersene.

that the binding of the 1st molecule of farncsyl pyrophosphate

to the enzyme is irreversibly connected to the binding of NAI>PI-I

(parallel pattern) and to the binding of the 2nd molecule of

farnesyl pyrophosphate (sf.raight lines in the plot of Fig. 11).

From this information we can conclude that the first partial reac-

tion has a ping-pong-type mcchankm (25, 36) (see Scheme I).

Confirmatory evidence for a ping-pang mechanism was obtained

from an initial velocity study of the first partial reaction. The

straight line obtained for the plot of the reciprocal of the initial

velocity against the reciprocal of farnesyl pyrophosphate con-

centration in the first partial reaction (Fig. 12) agrees with our

result on the initial velocity for the over-all reaction (Fig. 11).

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IO 20

dH IN (mM)-’

FIG. 11. The effect of a fixed variable concentration of farnesyl pyrophosphate and a variable concentration of NADPH on the initial velocity of the squalene synthetase reaction. The follow- ing were incubated at 37” in a volume of I ml : potassium phosphate baftfcr, pH 7.5, 50 mM; MgCL, 10 ml\l; glycerol, 0.3 ml; and protein, 20 fig. The concentrations of farnesyl pyrophosphate were: A--& 4.002 /.LM; a---a: 2.022 @I; O---o, 1.347 fiM; O-O, 0.991 PM. The concentrations of NADPH are indicated on the figure. The rate for each point was obtained from the slope of the line resulting from a plot of the squalene formed against incuba- tion times of 0, 1, 2, and 5 min. The procedure of assay for squa- lene was described previously (1, 20). Z~set, a replot of intercepts against the reciprocal of farnesyl pyrophosphate concentration.

-0.5 ' 0 0.5 1.0

‘IFARNESYL PYROPHOSPHATE INIpM)-’

FIG. 12. Double reciprocal plots of the initial velocity of pre- squalene pyrophosphatc formation against substrate concentra- tion. The following were incubated for 0, 1, 2, and 5 min at 37”: potassilml phosphate, pH 7.5, 50 mm1; magnesium chloride, 10 mM; glycerol, 0.3 ml; and purified sqllalene synthetase, 20pg of protein. The concentrations of farnesyl pyrophosphatc are indicated on the figure. The procedure of assay for presqualene pyrophosphate was report,ed previollsly (1, 20).

If the condensation of 2 moleculrs of laluesyl pyrophosphate

were sequential (both molecules of farncsyl pyrophosphate bound

to the enzyme surface before product is released), the double re-

ciprocal plot would generate a parabola (Equation 5). On the

ot,her hand, the equation for a ping-polg mechanism predicts straight lines (Equation I). More direct evidence for this mcch- anism would involve the isolation of an “ellzyIne-farncsyl” inter- mediate which is postulated for this reaction. In earlier studies Krishna et al. (5) reported the isolation of a GE intermediate

tightly bound to the enzyme. HOWXC~, it was not shoed that this group is covalently bound to squalcnc synthetase.

FIG. 13. A plot of the reciprocal of the initial velocity of the reduction of presqualene pyrophosphate catalyzed by squalene synthetase against the reciprocal of NADPH concentration. The following were incltbnted in a total volume of 0.5 ml at 37”: potas- sium phosphate buffer, pH 7.4, 50 mM; MgC12, 10 mM; glycerol, 0.15 ml; and purified squalene synthetase, 5 pg of protein. The concentrations of presqlialene pyrophosphate were: O--O, 1.678~~; O---O, 2.240,~~; n--A, 2.803~~; and O---O, 5.05 PM. The rate of reaction for each point was calculated from the slope of the line resulting from a plot of the squalene produced against incubations for 0, 1, 2, and 5 min. The assay procedure is reported under “lfxperimental Procedure.” Inset, the inset is a replot of intercepts and slopes against the reciprocal of presqua- lcne pyrophosphate concentration.

50 A IN lmM)-’

100

NADPH

FIG. 14. Effect of inorganic pyrophosphate on the rate of redllc- tion of presqaalene pyrophosphate to squalene. The following were incltbated in a volume of 0.5 ml at 37”: potassium phosphate buffer, pH 7.4, 50 m&I; MgClp, 10 mM; glycerol, 0.15 ml; and squa- lene synthetase, 5 pg. The concentration of presqualene pyro- phosphate was 1.02 PM. The concentrations of pyrophosphate were: l ~0,0.08 mM; n--a, 0.06 InM; O--Cl, 0.02 mM; and O--O, 0.00 mM. The rate for each point was obtained from the slope of the line resulting from a plot of sqllalene formed against incubation at 0, 2.5, and 5 min. The assay procedure is reported under “l<:xperimental Procedure.” I,rsel, a replot of intercepts and slopes against pyrophosphate concentration.

Initial velocity studies for the second llartial rcartion showed that it is a11 Ordered IG Tcr type. The possibilities existing for this reaction are (a) a completely random addition and release of products, and (b) a sequential ordered mechanism. If the addition of substrates or the release of ljroducts were random,

none of the products would give comprtitivc inhibition with an>

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I I ON 0.‘2 0’

NADP IN(mM) 2

1 4 6

PRESOUALENE PYROPHOSPHATE IN (i-‘M)-’

FIG. 15. The effect of fixed variable concentrations of NADP on the initial velocity of reduction of presqualene pyrophosphate to squalene. The following were incubated in a total volume of 0.5 ml at 37’: potassium phosphate buffer, pH 7.4, 50 mM; MgCL, 10 mu; glycerol, 0.15 ml; and protein, 5 ,~g. The concentration of NADPH was 0.0174 mM. The concentration of presqualene pyro- phosphate is indicated on the figure. The concentrations of NADP were: O---O, 0.186 mat; U----O, 0.069 mM; A-A, 0.035 rnM; O--O, 0.00 mM. The rate for each point was deter- mined from the slope of the line resulting from a plot of squalene formed against incubation of 0, 1, and 2.5 min. The assay pro- cedure is reported under “Experimental Procedure.” Inset, a replot of intercepts and slopes against NADP concentration.

0 01 NAOP IN ImM) 0 50 100

& IN (FM)-’

FIG. 16. The effect of fixed variable concentrations of NADP and variable concentrations of NADPH on the initial velocity of reduction of presqualene pyrophosphate to squalene. The fol- lowing were incubated in a total volume of 0.5 ml at 37”: potassium phosphate buffer, pH 7.4, 50 mn; MgC12, 10 rnx; glycerol, 0.15 ml; and purified squalene synthetase, 5/~g of protein. The concentra- tion of presqualene pyrophosphate was 2.802 P’M. The concentra- tions of NADPH are indicated on the figure. The concentrations of NADP were: O---O, 0.133 mM; A--A, 0.067 mM; U-O, 0.033 mM; and O---O, 0.00 mM. The rate for each point was ob- tained from the slope of the line resulting from a plot of the squa- lene formed against times of 0, 1, and 2.5 min. The assay pro- cedure is reported under “Experimental Procedure.” Inset, a replot of the slope against NADP concentration.

substrate. As we obtained an S-linear, competitive inhibition

when NADP was added as an inhibitor and NADPH was the

variable substrate, a random, or a partially random mechanism

for the addition of substrates, the release of products, or both,

must be discarded. Therefore, it is concluded that the second

partial reaction is sequential ordered.

When P (the first product to be released from the enzyme) is

present during the reaction, Equation 3 can be rearranged and

simplified to Equation 6, which predicts an S-linear, I-linear

noncompetitive inhibition when the first substrate is varied.

(6)

I When R (the last product to leave the enzyme) is present in the

incubation mixture, Equations 7 and 8 are obtained by rearrange-

ment and simplification of Equation 3.

FPP PP FPP PSQPP

-IL-l-L E-FPP E-F E-PSQPP

SCHEME I. Proposed mechanism for the formation of presqua- lene pyrophosphate. FPP, farnesyl pyrophosphate; PP, inorganic pyrophosphate, PSQPP, presqualene pyrophosphate; and E-F, farnesyl-enzyme.

TABLE II

Comparison of experimental and theoretical patterns of inhibition by products

Variable substrate

A. ................. A. ................ B ..................

NADPH ..........

NADPH ..........

Presqualene pyro- phosphate. ......

Fixed variab!e substrate Inhibitor Pattern of

inhibition

B (NS)a

B (NS) A (NS) Presqualene py-

rophosphate Presqualene py-

rophosphate

P NC.b R C.C R NC. Pyrophosphate NC.

NADP C.

NADPH NADP NC. -

a NS, the fixed substrate is not saturating.

b NC, noncompetitive inhibition. c c., competitive inhibition.

Since NADP and NADPH bind to the same form of enzyme

(competitive inhibition), NADPH must be the first substrat.e and NADP the last product to be released. Since NADPH is the first substrate to bind to the enzyme presqualene pyrophos- phate has to be the second substrate to bind.

Some of the possibilities that can be predicted by the use of Equations 6, 7, and 8, and the experimental results, are sum- marized in Table II. The results suggest a sequential ordered

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-cI OPP

I

CH3wCH20PP

RB %-

CH3GCH20PP

-H P

Rs hH

5H3 CH3 m

R=CH3-C=CH-GHz-CH&=CH-CH+42-

SCHEME II. Proposed mechanism for the first partial reaction, the conversion of farnesyl pyrophosphate to presqualene pyro- phosphate.

mechanism of addition and release of products with NL4DPH as the first substrate, followed by presqualene pyrophosphate (as the second substrate), and inorganic pyrophosphate as a first product followed by squalene and NADP.

Several mechanisms have been proposed for the formation of presqualene pyrophosphate (10-13). ill1 of these mechanisms involve the sequential addition of farnesyl pyrophosphate. Our results, and those of Dugan and Porter (24) for the pig liver squa- lene synthetase, point to a ping-pang type of reaction with the formation of a farnesyl-enzyme intermediate. A mechanism for the reaction that fits the kinetic data is presented in Scheme II. In the first reaction an SN2 displacement of the pyrophos- phate moiety by a nucleophilic group of the enzyme yields a farnesyl-squalene synthetase intermediate. The r system of C-2 of a 2nd molecule of farnesgl pyrophosphate then displaces the enzyme from C-l of farnesyl-enzyme. This displacement is helped by a second nucleophilic group of the enzyme. The over- all effect of this double displacement is retention of the configura- tion around C-l of both of the original molecules of farnesyl py- rophosphate. One or both of the nucleophilic groups on the enzyme could be a sulfhydryl group. This could explain the inhibitory action of N-ethglmaleimide and other -SH reagents. The next step is isomerization of the double bond as postulated by van Tamelen and Schwartz (16) with the displacement of one of the hydrogens of the C-l from the 1st farnesyl pyrophosphate

,111 H

CHzOPP

SCHEME III. Alternative mechanism for the first partial reac- tion, the conversion of farnesyl pyrophosphate to presquaIene pyrophosphate.

NADPH PSQP?

E-NADFH /NADPH+ /SO

KFsQFF- EwA3 F

E-NADP

/PP E-SO

‘NADP

SCHEME IV. Proposed mechanism for the reduction of presqua- lene pyrophosphate. PSQPP, presqualene pyrophosphate; PP, inorganic pyrophosphate; SQ, squalene; and E, squalcne syn- thetase.

molecule. The t.hird step would then be an intramolecular SN2 displacement of the enzyme by the newly formed R elect.rons to form a cyclopropnne riiig. The last step would involve the for- mation of a tram double bond by elimination of a proton on the tertiary carbon with the establishment of an olefinic bond at the original site and the formation of presqualene pyrophosphate. Alternatively a direct removal of I/b from III and an attack on the carbon atom would displace the B group from the enzyme and form the cgclopropane ring (Scheme III). Both mechanisms agree with the results of Cornforth et al. (37) who showed that only 1 farnesyl residue loses a llydrogen from the C-l position on conversion of 2 molecules of farnesyl pyrophosphate to squalene.

Our kinetic data for t,he second part,ial reaction of squalene synthesis are consistent with an ordered sequent,ial mechanism as presented in Scheme 1V. The chemistry of such a mechanism has been proposed by Edmond et al. (12). Their proposal in- volves the rearrangement of the cyclopropane ring lvith the migration of the pyrophosphate moiety to form a tautomeric cyclobutyl derivative. This reaction is then followed by a direct

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transfer of the hydride from KADPH and elimination of the pyrophosphate anion to form squalene.

Acknowledgment-We m-ish Do cspress our appreciation to Dr. A. 31. Coates of the Department of Chemistry, University of Illinois, for the generous gift, of synthetic presqualene alcohol.

REFER BNCES

1. QURI:SHI, A. A., B~~YTIA, E., & POI~TXIZ, J. W. (1973) b. Biol. Chem. 248, 1848-1855

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(1971) ‘J. &l. Chek. 246, 625G-6271 13. RILLING. H. C.. DALE PBULTER. C.. EPSTEIN. W. W.. & L~R-

SEN, Ii. (1971j J. Amer. Chem.‘S& 93, 1783:1785 ’ 14. ALTXIN, L. J., LOWERSICY, E. C., 8.~ RILLING, H. C. (1971) J.

Amer. Chem. Sot. 93, 1782-li83 15. COATES, R. M., & RO~IINSOS, W. H. (1971) J. Amer. Chem.

Sot. 93, 1785-1786

16. V‘YN TAMELEN, E. E., & SCHYI~I~TZ, M. A. (1971) J. Amer. Chem. Sot. 93, 1780-1782

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Enrique Beytia, Asaf A. Qureshi and John W. PorterSqualene Synthetase: III. MECHANISM OF THE REACTION

1973, 248:1856-1867.J. Biol. Chem. 

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