Effects of Site-directed Mutagenesis of the Highly Conserved ...

Vol. 267, No. 30,Issue of October 25, pp. 21873-21878,1992 Printed in U.S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of Site-directed Mutagenesis ofthe Highly Conserved Aspartate Residuesin Domain I1 of Farnesyl Diphosphate Synthase Activity* (Received for publication, April 27, 1992)

Pedro F. MarreroS, C. Dale Poulters, andPeter A. EdwardsSll From the $Departments of Biological Chemistry and Medicine, UCLA, Los Angeles, California90024 and the §Department of Chemistry, Uniuersity of Utah, Salt Lake City, Utah 841 12

Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthaseshow the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transferenzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the of role this second aspartaterich domain in rat FPPsynthase, we mutated the first or third aspartate to glutamate, expressed the wildtype and mutantenzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mgofhomogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parametersof the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: V,,, = 0.84 pmol/ ; diphosphate min/mg; G P P K , = 1.0 p ~ isopentenyl (IPP) K,,, = 2.7 p ~ Substitution . of glutamate for the first aspartate (EDYLD) decreased the VmaXby over 90-fold. TheK , for IPP increased, whereas the K , for GPP remained the same in thisD243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in theD247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.

generically referred to as prenyltransferases. One group of prenyltransferases, which includes FPP synthase, hexaprenyl diphosphate synthase, and geranylgeranyl diphosphate synthase (3-6), catalyzes the irreversible 1’-4 condensation of homoallylic isoprene units to an allylic diphosphate primer (2). Other prenyltransferases, such as squalene synthase (7, 8) and phytoene synthase(crtB) (9, lo), are bifunctional enzymes. They catalyze the cl’-2-3 condensation of two allylic diphosphate substrates to form presqualene diphosphate or prephytoene diphosphate, respectively, followed by a 1’-1 rearrangement of the cyclopropylcarbinyl intermediates. A third group of prenyl transferases are involved inthe transfer of allylic units to various non-terpene acceptors. Such prenylating enzymes include para-hydroxybenzoate-polyprenyl transferase, tRNA-dimethylallyl transferase (MOD5), adenosine monophosphate-dimethylallyl transferase(IPT: TiBo542, TiT37, TiAch5), or protein:farnesyl transferase (see Ref. 11, and references therein). Thus, allprenyl transferases utilize an allylic isoprene unit as one substrate to alkylate a variety of compounds containing electron-rich moieties. Comparison of the amino acid sequences encoding FPP synthase from four species (Homosapiens,Saccharomyces cereuisiae, Rattusrattus, and E. coli) with the amino acid sequences from the related enzymes hexaprenyl diphosphate synthase (from S. cereuisiae) and geranylgeranyl diphosphate synthase (from Neurospora crassa)shows the presence of two aspartate-rich conserved domains, (I, L, or V)XDDXXD, where X encodes any amino acid (3). Based on the presence of these two aspartate-rich conserved domains, we have previously proposed that they may be involved in enzymecatalFarnesyl diphosphate (FPP)’ synthase catalyzes the se- ysis, perhaps by binding the allylic and homoallylic substrates quential 1’-4 condensation of isopentenyl diphosphate (IPP) (394). In order to test this hypothesis, we separately mutated 2 with the allylic diphosphate, dimethylallyl diphosphate, and aspartates in domain I1of rat FPP synthaseand overexthen with theresultant geranyl diphosphate (GPP).The ultimate product of these two reactions, FPP, is utilized in pressed the mutant and thewild-type enzymes in E. coli. We the synthesis of squalene, farnesylated proteins, dolichols, demonstrate that thekinetic parameters of the purified wildcoenzyme Q, geranylgeranyl diphosphate, and the isoprenoid type rat recombinant FPP synthase aresimilar to the purified human, pig, and avian enzymes. The kinetic parameters of moiety of heme a (1). FPP synthase is but one of a large number of enzymes the mutant rat FPP synthase enzymes suggest that the first, but not the third, aspartate in domain I1 is critical for the * This work was supported by National Institutesof Health Grants conversion of GPP to FPP. Replacement of the first aspartate HL 30568 and GM 21326 and by the Laubisch Fund. The costs of of this domain with glutamate results in a 90-fold decrease in publication of this article were defrayed in part by the payment of V,,, and an increase in K,,, for IPP. page charges. This article must therefore be hereby marked “aduertisement” in accordance with18U.S.C. Section 1734 solely to indicate this fact. T T o whom correspondence shouldbe addressed Dept.of Biological Chemistry, University of California, Los Angeles, CA 90024. ’ The abbreviations usedare: FPP, farnesyl diphosphate; GPP, geranyl diphosphate;IPP, isopentenyl diphosphate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; kb, kilobase pair(s).

EXPERIMENTALPROCEDURES

Materials and General Pro~edures-[l-’~C]IPP(52.7 Ci/mol) was purchased from Amersham Corp.MicrocrystallineDE52ion exchange cellulose was obtained from Whatman.Oligonucleotides were synthesized by the pbosphoramidite method (12) by Dr. Dohn Glitz (UCLA) ona Du Pont/Vega Coder 300 DNA synthesizer. Radioactivity was measured in Bio-Safe I1 scintillation media (Research Prod-

21873

21874

Domain 11of FPP Synthase

ucts International Corp.) with a Beckman model LS 6000SC liquid scintillation spectrometer. Proteinswere analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with the discontinuous buffer of Laemmli (13).The gels were stained withCoomassie Brilliant Blue R. Protein concentrationswere determined by the methodof Bradford (14) using bovine serum albumin asa standard. Recombinant DNA Methods and Reagents-Enzymes used in plasmid constructions were obtained from Bethesda Research Laboratories. Preparation of plasmid DNA, restriction enzyme digestion, and agarose gel electrophoresis were carried out by standard procedures as describedby Maniatiset al. (15).DNA sequencing of cDNA subclones was performed by the dideoxy chain termination method ECORI NdeI I of Sanger et al. (16), using either M13 universal primers or specific 1) P C R 21 XhoI/EcoRI oligonucleotides. Polymerase chain reactions (PCR) were conducted pBlusecript 1 with theGeneAmp Kit (U. S. Biochemical Corp.). The plasmid CR39, XhoI/EcoRI 3 ) T4 ligase which contains the F P P synthase from rat liver, has been isolated previously (17-19). sequence Strains, Media, and Transformations-E. coli strain XL-blue was used for allplasmid manipulations. E. coli strain JMlOl (21) was used for the expression of recombinant FPP synthase. Competent S a cells were prepared, stored, and transformed by established proceI e 1 I I dures (15). For expression studies, cultures of JMlOl transformants MUTANTS e S.S. t 5 ' ' 1 were grown on a supplemented M9 minimal medium (M9 + CAGM) pFPPSl4 (4.03 Kb) containing thefollowing: M9 salts and trace elements (15), casamino I acids (176, w/v), glucose (0.26%, w/v), magnesium sulfate (0.3 g/liter), calcium chloride (4 mg/liter), thiamine hydrochloride (5.4 mg/liter), 1 ) SalI/NdeI and either ferrous chloride (5.4 mg/liter) or ferrous ammonium sulfate pARC306N ____) (10.9 mg/liter). Salr/Ndel SalI/NdeI 2) T4 ligase Construction of aReading-frameCassette andtheProkaryotic Expression Vector for the FPP Synthase cDNA-The strategy and the primers used in the construction of the reading-frame cassette for the F P P synthase by PCR is explained in Fig. 1. The strategywas pFPPS19 (D243E) pFPPS16 (3.59 Kb) similar to that reported by Street etal. (21) in the construction of the pFPPS20 (D247E) reading-frame cassettefor the yeastIPP isomerase. Four new restriction sites were introduced into the rat liver FPP synthase cDNA by FIG. 1. Construction of a reading-frame cassette and a proPCR-mediated,site-directed mutagenesis;two of those sites were karyotic expression vector for wild-type (DDYLD) and muintroduced into the region immediately upstream of the translation tant (D243E, D247E) FPP synthase. Four new restriction sites initiation codon, and the other two were introduced into the region were introduced by PCR mediated mutagenesis in the 1.159-kb rat immediately downstreamfrom the translation terminatorcodon (Fig. F P P syrkhase cDNA (thenew nucleotides introduced in thesequence 1). Theupstreamprimer was 28 nucleotides inlength,andthe areindicated by asterisks). ThePCRamplificationproduct was structure was basedon positions -22 to +6 (sense strand) of the digested with XhoI/EcoRI, and the 1.097-kb fragment was ligated published sequence for the rat FPP synthase cDNA (19). Mutations into the 2.933-kb XhoI/EcoRI fragment of pBluescript(SK+) to give introduced by this primer were a two-base alterationat positions -20 pFPPS14. pFPPS14 wasused to sequence the PCR amplification and -16 (C to A and T to C, respectively), which created the EcoRI product and to generate rat FPP synthase mutants D243E restriction site, and a three-base mutation a t positions -3, -2, and (pFPPS17), andD247E (pFPPS18). Thewild-type and mutant read-1 (A to C, G to A, and A to T,respectively), which created the NdeI ing-frame cassettes were removed from pFPPS14, 17, and 18, as a restriction site. The downstream primerwas 27 nucleotides in length, NdeIISalI fragment. The bacterial expression vectors pFPPS16, 19, and the structure was based on positions1080-1054 (antisense strand) and 20 were constructed by ligating the1.070-kb NdeIISalI fragment of the FPPsynthase. Mutations introducedby this primer were a 2- derived from pFPPS14, 17, and 18, respectively, to the 2.52-kb NdeI/ base alteration a t positions 1064 and 1069 (sense strand numeration, Sal1 fragment of pARC306N. G to C and T to G, respectively), which created the Sal1 restriction site, and another2-base mutation at positions1072 and 1074 (C toG and T to G),which created the XhoI restriction site. Template cDNA ATCTGGAA-3' (C to A sense strand substitution at position 756) a t position number 243 for the PCRwas prepared from CR39-1.2 (17) by isolation of a 1.159- for aspartatetoglutamatesubstitution kb fragment after restriction endonuclease digestion with EcoRI.The (D243E mutant; EDYLD; pFPPS17), and 5"GGGTCTCCAAAGAPCR reaction mixture contained thefollowing: 6 mM MgSO,, 250 ng GCTCAAGGTAGTCGTC-3' (T to G sense strand substitution a t at position of template cDNA, 1 Gg of each primer, 200 FM each dGTP, dATP, position 768) for aspartatetoglutamatesubstitution dTTP, and dCTP, and 2.5 units of TaqDNApolymerase. This number 247 (D247E mutant; DDYLE; pFPPS18). Base changes in mixture was taken throughfive cycles with thefollowing temperature thecDNA were confirmed by dideoxy sequencing using specific changes: denaturation (2 min, 95 "C), annealing (3 min, 37 "C), and primers (17-19). The NdeI/SulI 1.070-kb fragments containing the expressionvector extension (2 min, 75 "C). After the fifth cycle, Taq polymerase was FPP synthasemutants were insertedintothe above. The new expression vectors: inactivated by phenol extraction, and the DNA was precipitated with pARC306N, as described ethanol. The PCR product mixture was digested with EcoRI and XhoI pFPPS19 (D243E), and pFPPS2O (D247E) were used to transform and thenligated into the EcoRI and XhoI sites of pBluescript SK(+) E. coli strain JM101. The insert from pFPPS19 was sequenced to (pFPPS14, 4.030 kb). The 1.070-kb FPP synthase coding region was ensure that no unwanted mutations had been introduced into the isolated from pFPPS14 after NdeIISalI digestion and ligated to the open reading frame during the PCR step. Purification of Wild and Mutant Recombinant FPP Synthase2.52-kb NdeIISalIfragment of thebacterial expressionvector pFPPS19, or Typically, 150-ml cultures of JMlOl/pFPPS16, pARC306N (Fig. 1). The resulting F P P synthase expression vector (pFPPS16, 3.59 kb) was used to transform E. coli strain JM101, and pFPPS2O were grown on M9 + CAGM containing 100 Fg/ml ampithetransformants were used for expression studiesand enzyme cillin, in a 1-liter flask a t a 37 "C with vigorous aeration. The cells were harvested by centrifugation (5,000 X g, 10 min), and the cell isolation. Site-directed Mutagenesis of FPP Synthase-cDNA mutants for paste was resuspended in buffer A (10 mM potassium phosphate, 10 rat FPPsynthase with a substituted amino acid in the aspartate-rich mM p-mercaptoethanol, 1 mM EDTA, pH 7.0), to a final density of 1.5 g/10 ml. The cells were disrupted by sonication, while beingcooled domain I1 were constructed by oligonucleotide-directed in vitro mutagenesis with an Amersham mutation kit. The single strand template in ice water. Sonication was carried out for three cycles of 30 s, the extract being allowed to cool between each cycle for another 30 s. All wasgenerated from pFPPS14.Thesyntheticmutagenicprimers, containing a single mismatch with the wild-type cDNA, used for the subsequent procedures were performed a t 4 "C. The extract was mutantconstruction were: 5'-AGATCAAGGTAGTC7TCCTGG- clarifiedby centrifugation (15,000 X g, 15 min), and total protein

II I

T

I

-

Domain II of FPP Synthase concentration and FPPsynthase activity were determined. The protein precipitating between 35-70% ammonium sulfate was collected and the pellet suspended in buffer A to a final volume of 2 ml. This solution was dialyzed for 18 h againsttwo 1-liter changesof buffer A. After centrifugation a t 15,000 X g for 10 minthe solution was diluted to a protein concentration of about 10 mg/ml. The diluted extract was pumped onto a 20 X 2.5-cm DE52 column (flow rate 2 ml/min), which had been equilibrated with buffer A. The column was washed with buffer A until the absorbance of the eluate had returned to baseline value. A linear gradient (400 ml) of10-90 mM potassium phosphate (pH 7.0) containing 1 mM EDTA and 10 mM P-mercaptoethanol was applied to thecolumn. A single peak of FPP synthase activity emerged after about150 ml of the gradient had been collected. Fractions with FPP synthase activity were combined (total volume about 90 ml),concentrated to afinal volume of1.2 ml with a Centriprep 30 (Amicon), and stored frozen a t -70 "C. Assay of Farnesyl diphosphate Synthase-The enzyme was assayed essentially by the method of Holloway and Popjak (22) as detailed recently (23). Routine assays contained (in 0.11-ml incubation) 40 pM [1-"Clisopentenyl diphosphate, specific activity 1 Ci/mol, and 20 p~ geranyl diphosphate. For kinetic studies with purified enzymes, the 0.11-ml incubations contained10 mM Hepes buffer, pH 7.2,l mM MgC11, 1mM dithiothreitol, and either 3.7 ng of wild-type enzyme, 4 ng of the D247E mutant, or 5 pg of the D243E mutant. Assays were carried out a t 37 "C for 5 min with varied substrate concentrations as indicated in the figures. In these assays [l-'4C]isopentenyldiphosphate with a specific activity of 4 Ci/mol (Fig. 4, A and €3) or 52.7 Ci/mol (Fig. 4C) was used. The duplicate assays were terminated by acid hydrolysis, and the hexane-soluble radioactivity determined as described (23). Variations of duplicate assays were less than 10%. In all cases less than 10% of the substratewas converted to product. A separate blank assay containing no geranyl diphosphate was performed for each assay utilizing a different concentration of [l-"C] IPP.

21875

Expression of Wild-type and Mutant Rat liver FPP Synthase in E. coli Examination of cell-free extracts prepared from cultures carrying prokaryotic expression vectors: pFPPS16 (Asp243Asp-Tyr-Leu-Aspz4' wild-type FPP synthase),pFPPS19 (D243E mutant FPP synthase), and pFPPS2O (D247E mutant FPP synthase) by SDS-PAGE (Fig. 3, lanes 3,5, and 7, respectively) showed, in each case, a protein band that migrated with an apparent molecular mass of39 kDa. This protein was not present in cell-free extracts prepared from cells transformed with the expression vector pARC306N alone (Fig.3, lane 2). The apparent molecular mass of39 kDa determined here for rat liver FPP synthase is consistent with the predicted molecular mass calculated from the synthase cDNA nucleotide sequence (17-19). In addition, both mutant and wild-type FPP synthase were recognizedon immunoblots by antibodies raised against an FPPsynthase fusion protein (23) (data not shown). To determine whether the expressed protein was functional, we performed in vitro FPP synthase assays and confirmed that the product formed was farnesyl diphosphate. Analysis of the hexane-soluble products after acid hydrolysisfrom an in vitro assay using a cell-free extract that contained wild-type enzyme showed twopeaks of radioactivity on thin layer chromatogram which co-migrated with farnesol and nerolidol standards in the expected ratio of 1:4 (24) (data notshown).

Purification and Characterization of Recombinant Wild-type and Mutant Rat Liver FPP Synthase Wild-type and mutant FPP synthases were purified from RESULTS AND DISCUSSION crude cell-free extracts by ammonium sulfate precipitation Fig. 2 shows the alignments of the aspartate-rich domain I followed byion exchange chromatography on DEAE-cellulose. and domain I1 of different prenyl diphosphate synthases ((I, This procedure yielded proteins that gave a single band on L, or V)XDDXXD) (3, 4). All these enzymes are known to SDS-PAGE and were judged to be greater than 95% pure bind both homoallylic and allylic diphosphate substrates. It (Fig. 3, lane 4 for wild-type, lane 6 for D243E mutant, and is apparent that these enzymes share other conserved amino lane 8 for D247E mutant). Table I shows the yields, specific acids in addition to the aspartatedomain. These include I or activities, and purification factors for thethree enzymes. L, X4L to the amino side and RRG to the carboxyl side of Wild-type rat liver FPP synthase was overexpressed and domain I and I or L, GXzFQ to the amino side of domain 11, resulted in at least a 360-fold higher specific activity than the where X indicates any amino acid (Fig. 2, bold type). endogenousE. coli FPP synthase activity, and more than 30% In order to determine whether the conserved aspartates of the E. coli soluble protein was rat liver FPP synthase, as played a significant role in binding either the allylic or the homoallylic substrate or in the catalytic activity of the rat FPP synthase the normal and mutantenzymes, in which the glutamate had replaced an aspartate, were overexpressed in E. coli, purified, and assayed for enzymeactivity. (A)

I L L (S.C.) L HPS(S.c.) I GGPPS(N.c.) L

FPPS(E.c.) '' (R.r) (H.S.) "

"

DOMAIN I H Q Q Q H H

A A A A T T

Y F F Y A A

S F F F S S

L L L L L L

IH D D L P A M D D D D L R R G V L D D . . I M D S S Y T R R G V A D D . . I M D S S Y T R R C V A D D . M . M D K S IT R R G L H D D . .V E D N S V L R R C V E D N S V L R R C L V D D .

39 kDa

-+

.

I (6)

DOMAIN 11

FPPS(E.c.) I C L A F Q V Q D D IL D V V G D T A T L C K (H.s.,R.r.) M C E F F Q I Q D D Y L D L F G D P S V T G K .' (S.C.) L C E Y F Q IQ D D Y L D C F G T P E Q IC K HPS (S.C.) L C 1C F Q L V D D M L D F T V S G K D L C K GGPPS(N.c.) I C L I F Q I A D D Y H N L W N R E Y T A N K "

FIG.2. Alignment of the aspartate-rich domains I and I1 for different prenyltransferases. The optimal amino acid alignment between domain I or domain I1of farnesyl diphosphate synthase (FPPS) from four different species with hexaprenyl diphosphate synthase ( H P S )and geranylgeranyl diphosphate synthase (GGPS) is shown (sequences cited in Refs. 3-6). Highly conserved amino acids are shown in boldface.E.c.,E. coli; H.s., H. sapiens; R.r., R. ratus; S.C., S. cerevisiae; N.c., N . crassa).E. coli FPP synthase has 2 extra amino acids between the second and the third aspartate.

"

I

2

3

4

5

6

7

8

FIG.3. Expression and purification of wild-type and mutant rat FPP synthase recombinant proteinsas monitored by SDS/PAGE. 100 pg of protein from crude cell-free extracts (lanes 2, 3, 5 , and 7) or after the DEAE-52 purification step (lanes 4, 6, and 8) were electrophoresed on a 10% polyacrylamide gel and stained with Coomassie Blue. Lane 1, molecular weight standards; lane 2, JM101/pARC306N lanes 3 and 4, JMlOl/pFPPS16 (wt); lanes 5 and 6, JMlOl/pFPPS19 (D243E); lanes 7 and 8, JMlOl/pFPPS20 (D247E). The position of the 39-kDa FPS protein is indicated.

Domain 11of FPP Synthase

21876 TABLE I

Activity of rat FPP synthase expressed inE. coli Wild-type and mutant enzymes were expressed in E. coli. The activities of the enzymes were determined in the crude soluble extracts and afterpurification on DEAE-52. One gram of wet cells from each clone provided approximately 120 mg of soluble protein and 12 mg of homogeneous enzyme in 30% yield with a 2.5-fold increase inspecific activity. (Mmol/min/mg) Purification activity Specific step Crude extract DEAE-52 1.5

I

E. coli

1.4 & 1.0 X 1.0 ND'

DDYLD

EDYLD

DDYLE

7.2 0.50

X X

0.83 1.2

lo-'

1.0

/

2.0

0.5

l/[GPP]

1.0

l/[IPP]

-

The mean for four different E. coli extracts.

'No determination.

judged by the 2-fold increase in specific activity after purification to apparenthomogeneity on DEAE-cellulose (Table I; Fig. 3). The expression, yield, and purification factors obtained for both FPP synthase mutants, and thewild-type enzyme were similar (Table I). Mutation of the third aspartate in domain I1toglutamate (D247E) didnotresultinanysignificant change in enzyme specific activity (Table I). However, the specific activity for the D243E mutant was reduced by approximately 90-fold (Table I). This latter activity was more than &fold higher than theendogenous E. coli FPP synthase activity in crude bacterial lysates under the conditions of the assay. The molecular masses of the homogeneous wild-type and D243E mutant FPP synthase proteins under non-denaturing conditions were estimated by non-equilibrium ultracentrifugation in a sucrose gradient (5-20%, w/v). A single symmetrical peak of protein corresponding to a mass of 80-85 kDa was seen for both (data not shown), consistent withhomodimeric structures.

5 r

5 r

,

I II Iil

Iv ' I

2.0

1.0

/I//

1.0

0.5

l/[GPP]

Kinetic Studies Fig. 4showsdouble-reciprocal plots of initial velocities versus concentration with the concentrationof one substrate being varied while the other remained constantfor the wildtype(DDYLD) (Fig. 4A), D247E mutant (Fig. 4B),and D243E mutant (Fig. 4C) enzymes. Wild-type Rat Liver FPP Synthase (Fig. 4A)"At the concentrations tested (see Fig. 4, legend), the plotsof l / v against l/[S] gave straight lines, which intersected at points to the left of the y axis and below the abscissa. Thesedataare compatible with an ordered sequential reaction mechanism previously deduced for the pig liver (22)andavian liver enzymes (25, 26). Replots of the slopes and intercepts for the LineweaverBurke plots shown in Fig. 4, against the reciprocals of unvariedsubstrateconcentrations, gave straight lines (data not shown) that were used to calculate the kinetic parameters that are summarizedin Table I1 (27). Kinetic constantsfor rat liver FPP synthase have not been previously reported. V,,, for the synthesis of FPP from G P P and IPP by the wild-type rat liver enzyme shown here (0.84 pmol/min/mg) is similar to the values reported for human and avian liver FPP synthases (28, 29). The K,,, values calculated for both substrates (K,(GPP)= 1.0 p~ and K,(IPP)= 2.7 PM) are also similar to thosepreviously reported for FPP synthase from human liver: 0.44 and 0.94 pM, respectively (28); avianliver, 0.5 p~ for each substrate (29); pig liver, 4.3 and 1.5 pM (22) or 0.58 and 0.31 pM (30); E. coli, 8.7 and 4.7 p~ (31); and yeast 14 and 4 p M , respectively (32). The 0 2 4 7 3 Mutant (Fig. 4B)"Substitution of the third

1

3

3

0.05

0.10

l/[GPP)

1mJI

FIG. 4. Lineweaver-Burk plots for wild-type ( A ) and mutant D247E ( B ) or D243E ( C ) FPP synthase. Plots of l / u (pmol-'. min. mg), against the reciprocal of the concentrations( p c " ' ) as

of the varied substrate a t different fixed concentrations of the second substrate are shown. Graphs on left show varied concentrations of geranyl diphosphate (GPP) atdifferent fixed concentrations (in p M ) of isopentenyl diphosphate (IPP). A: line i, 1.25; ii, 1.66; iii, 2.5; iu, 3.3. B: i, 1.0; ii, 1.25; iii, 1.66; iu, 2.5. C: i, 8; ii, 16.6; iii, 25; iu, 33; u, 40. On the right are shown varied concentrations of I P P a tdifferent fixed concentrations ( p ~ of) GPP. A: i, 0.66; ii, 0.8; iii; 1.0; iu, 1.66; u, 2.5. B: i, 0.66; ii, 0.8; iii, 1.0; iu, 1.66. C: i, 0.5; ii, 1.0; iii, 1.66 iu, 2.0.

TABLEI1 Kinetic parametersof wild type and mutant ratFPP synthase proteins V,.,, Km(GPP), and Km(lpP) were evaluated from slope and intercept d o t s of the Lineweaver-Burk plots inFig. 4. pnol min" mg"

9

Wild type D247E D243E

70

0.84 1.95 X 10-3

PM

2.7 2.9

PM

1.0 1.1

1.1

Domain II of FPP Synthase

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dramatically decrease k4, the rate of dissociation of IPP from the E . GPP IPP complex, the expression simplifies to Km(1pp) k4/k3,the dissociation constant for IPP from the ternary = 5 p~ for a metal-free E . GPP. IPPternary complex. KD(Ipp) complex of the avian liver enzyme and is only slightly lower complex. If KD(lpp) is similar for for theE. Mg2+IPP. Mg2+IPP the avian and ratliver enzymes, the net effect of the mutation on IPP binding at the homoallylic site is slight. If, on the other hand, the 90-fold decrease for the D243E mutant resulted from an additional decrease in the rate of product release (41,Km(IPP) kdk4 + k5)/k3k5 and K m ( G P P ) ks/kl. As discussed for the previous case, a concomitant decrease in kl is required to maintain a similar value for K,(GPP) in the wild-type and D243E enzymes. To offset the decrease in &, the ratio k4 + k 5 / 4 k 5 must increase by approximately for the D243E 1,600-foldto give a 23-fold increase in K,,,(IPP) the change mutant. Assuming kgwas not substantially altered, must arise from a decrease in kS (the on rate for IPP), an increase in k4 (the off-rate for IPP from the ternary complex), or a combination of the two. This would suggest that D243 is involved in IPP binding, perhapsthrough chelation of a magnesium bound to the diphosphate moiety. However, this ukl[GPP] k~[IpPl k ks explanation requires that the D243 mutant have a reduced E,E.GPP~E.GPP.IPP 4 E.PP,.FPP + E affinity for IPP in the ternary complex with GPP and an k, k4 (1) increased affinity for FPP after catalysis. It is unclear how where the mutation could generate opposite affinities for IPP and FPP by an interaction with the same diphosphate moiety. Of the two scenarios, we favor an effect at k, rather than ks. The results with the D243E mutant suggest that the conserved domain I1 motifs in prenyltransferases are important for catalysis. Our kinetic analysis and the conservation of domain I1 among a wide variety of prenyltransferases, including enzymes that do not utilize IPP, suggest that domain I1 is the allylic binding domain. The aspartate atposition 243 is clearly important. Possible roles include specific acid catalysis Release of products is rate-limiting for condensation of IPP by protonation of the diphosphate moiety in GPP or binding and GPP, where kg 50ks, and V,,, &[Et].At saturating througha magnesium-diphosphate bridge. Unfortunately, concentrations of both substrates,the 90-fold decrease in V,,, there is insufficient information about the three-dimensional seen for the D243E mutant could result from a -3,500-fold architecture of the active site for us to provide a structural decrease in the rateof the chemical step ( k 6 ) ,with a concom- interpretation of our data. itant change in the rate-limiting step, or from an additional We were surprised to discover that the kinetic constants 90-fold reduction in the product release step ( k 6 ) . for the D247E mutant were virtually identical to those of the The chemical mechanism for 1'-4 condensation is an elec- wild-type enzyme. Aspartate is highly conserved at this locatrophilic alkylation of the double bond in IPP by the carbo- tionin l'-4 prenyltransferases. However, the recently recation derived from the allylic substrate, followed by elimi- ported putative amino acid sequence of geranylgeranyl dination of a protonfrom the original C(2) position in IPP (33). phosphatesynthase from N . crassa has an asparagine at This part of the catalytic cycle is represented by k,, which position 247 (34). In addition, the prenyltransferases which reflects the rate for rupture of the carbon-oxygen in theallylic catalyze alkylation of the amino moiety in AMP by dimethysubstrate togenerate the electrophilic allylic cation. If Kg were lallyl diphosphate during cytokinin biosynthesis contain leurate-limiting, the 3,500-fold decrease suggests that D243 is cine or isoleucine at the equivalent location of their domain important for formation of the carbocation, and its function I1 motifs (34). is impaired as the tether to the side-chain carboxylate is In conclusion, this is the first reported purification of rat reduced by a methylene. FPP synthase. Recombinant wild-type and mutant proteins The Michaelis constants for GPP in the wild-type and were purified in one step on DEAE-cellulose. The wild-type given enzyme has physical and catalytic properties that are similar D243E enzymes are similar. The expression for Km(GpPj in Equation 3 can be simplified to K,,,(GPP) ks/kl for wild- to those of the human and avian liver FPP synthase. The type (k6