PlantPhysiol.(1988) 88, 315-320 0032-0889/88/88/0315/06/$0 1.

00/0

in Production Phalarisaquatica N,N-Dimethyltryptamine Seedlings
A MATHEMATICALMODEL FOR ITS SYNTHESIS
Receivedfor publicationDecember31, 1987and in revisedform April26, 1988 JOSEPHP. G. MACK, DAWN P. MULVENA, AND MICHAEL SLAYTOR*

Maryland21228 of Chemistry Department,University MarylandBaltimoreCounty,Catonsville, of (J.P.G.M.),and Departmentof Biochemistry,University Sydney, Sydney,N.S. W.,2006, Australia(D.P.M., M.S.)
ABSTRACI

of The activitiesof threeenzymesandthe concentration intermediates (DMT) fromendoginvolved the synthesisof N,N-dimethyltryptamine in enous tryptophan(TRP) have been measuredin vitro in seedlings of Commercial over 16 days afterplantPhalarisaquaticaL. cv Australian ing. The activitiesof tryptophan decarboxylase the two N-methyland transferasesincreasedrapidlyto maximalrates of substrateconversion at day 5 of 95, 1000, and 2200 micromolesper hour per milliliter, respectively. Afterthese maximalrates, the activitiesdecreasedrapidly. The concentration intermediates of increasedrapidlyfrom zero in the at seeds to maximalvaluesof 25 and53 micromolar day 5 for tryptamine 1000 micromolar day at (T) andN-methyltryptamine (MT), respectively, of at 6 for TRP, and650 micromolar day 8 for DMT. The concentration DMT and of all the intermediates its synthesisdeclinedrapidlyafter in the maximal value had been reached. A mathematicalmodel of the pathwayfromTRP to DMT using these enzymescorrectlypredictsthe is of whoseconcentration deterconcentrations T andMT, intermediates minedonly by the pathway,and confirmsthat these three enzymesare responsible the in vivosynthesisof DMT. Kineticstudiesare reported for uses pyridoxalphosphate in vivo synthesis of DMT. for these enzymes.Tryptophan decarboxylase = KJFALP (PALP) as a coenzymeand has the followingkineticconstants: the Thispaperreports activitiesof enzymesand concentrations

grasses, particularlyunpalatabilityto sheep, which has been linked in P. aquaticato the graminecontent (4), a considerable amount is known of the factorsinvolvedin alkaloidproduction. For example,genetic evidence in P. arundinaceaindicatesthat gramine synthesis is independent of DMT' and 5MeODMT formation(1 1). This is furthersupportedby the findingthat the N-methyltransferase activitiesinvolved in graminesynthesisare different from those involved in the synthesis of DMT and 5MeODMT(13). Feeding experimentswith P. aquatica have establishedthe biosynthesisand turnoverof DMT and SMeODMT(1), while of the biosynthesis graminehasbeen studiedin the closelyrelated reedcanarygrass,P. arundinacea Enzymesfrom P. aquatica (9). that catalyzethe in vitroformationof DMT have been partially characterized, namely TDase (2) and the two SAM-dependent N-methyltransferases, PIMaseand SIMase(10), which methylate T and MT, respectively. Becauseof the smallnumberof enzymes involved,the synthesisof DMT from TRP offersthe possibility an of completelycharacterizing alkaloid-synthesizing systemand of testingthe hypothesisthat the enzymesare responsible the for

randomwith formationof both dead end complexes.Secondaryindolethylamine-N-methyltransferase methylatesbothMT and5-methoxy-Nmethyltryptamine (5MeOMT).The kineticconstantsforthe methylation MATERIALS AND METHODS of MT are:KMT = 40 ? 6, KSAM = 55 ? 15, KDMT = 60, KSAH = 4.3 ? with unity interaction factors.The kineticconstantsfor 0.4 micromolar Labeled Compoundsand Chemicals. L-['4C-Methylene]trypthe conversion of 5MeOMT to 5-methoxy-N,N-dimethyltryptaminetophan (["4C]TRP) ['4C-methyl]SAM and (['4C]SAM) were pur(5MeODMT)are KsMeOMT = 40 ? 10, KSAM = 90 ? 40, andKSAH = 2.9 chased from AmershamAustraliaPty Ltd. PALP and the sub? 0.3 micromolarwith unity interactionfactors, except for SAM- strateswere obtainedfrom SigmaChemicalCo., St Louis, MO. 5MeODMT= 2.0 ? 0.9 andSAH-5MeOMT= 0.45 ? 0.25. The kinetic [I4C]SAM usedwas assayedfor SAH contentby chromatography are = constantsfor primary indolethylamine N-methyltransferase RT. 20, on Whatman 3MM paper using butanol/acetic acid/water
= KSmAM 40, KMT = 450 micromolar with the substrates binding inde-

of reactantsof the DMT synthesizingpathwayas a function of age of P. aquaticaseedlingsfor the first 16 d of seedlinggrowth. nine (SAM) as substrate; S-adenosylhomocysteine (SAH) is assumedto These data togetherwith the kinetic constants of the enzymes be the product.The mechanismof secondaryindolethylamine-N-meth- have been used to model the DMT synthesizingsystem matheyltransferase, determined initialvelocitystudies,is rapidequilibrium maticallyduringthis initial 16 d of seedlinggrowth. by

2.5 micromolar, KIRP = 200 micromolar, KIT = 5 millimolar, and use The N-methyltransferases S-adenosylmethioKMT = 4 millimolar.

pendently.

(12:3:5).The nucleosideswere detectedwith Cd-ninhydrin(3), elutedwith methanol,and quantitated absorbance 500 nm. at by The SAM used in the assay contained0.024% SAH, which, at the highestconcentrationof SAM used (200 ztM), gives an SAH

'Abbreviations: DMT, N,N-dimethyltryptamine; 5MeODMT, 5methoxy-N,N-dimethyltryptamine; TDase, tryptophan decarboxylase; N-methSAM,S-adenosylmethionine; PIMase,primary indolethylamine Phalarisspp., notably Phalarisaquaticaand Phalarisarundi- yltransferase; SIMase,secondaryindolethylamine N-methyltransferase. MT, TRP, tryptophan; PALP,pyrnacea, are major pasture grasses in many parts of the world. T, tryptamine; N-methyltryptamine; SAH, S-adenosylhomocysteine; with respectto. wrt, Because there are undesirable features associated with these idoxalphosphate;
315

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MACK ET AL.

PlantPhysiol.Vol. 88, 1988

concentration the assayof 0.05 aM. This is small comparedto in for KSAH the enzymesand the concentrationof SAH used in the inhibitorbindingstudies. Preparation EnzymeExtracts. Seedlingsof Phalaris aquaof tica L. cv AustralianCommercialwere grown as describedpreviously (10). Whole seedlings(i.e. all plant materialincluding the testa for pre-emergent seedlings)(5 g) were homogenizedin the assaybuffer(1:2, v/w) with sand. An additional1%(v/v) of mercaptoethanol addedto the homogenate,which was then was centrifugedat 5000g for 5 min. The supernatant(1 mL) was passedthrougha SephadexG-25 (fine) column (1.5 x 6 cm) to separatethe proteinsfrom endogenousalkaloidsand other low mol wt compounds. The excluded protein fraction (3 mL), referredto as the enzyme extract, was collected and used for enzyme assays.It behavedsimilarlywith respectto linearitywith time and enzyme concentrationand gave the same kinetic constants as the original preparation.No differences in kinetic constantswereseen for preparations fromleavesor whole plants. The kineticswere investigatedat constant pH and the effect of hydrogenion concentration was not investigated. EnzymeAssays. TDase. The assay mixture consisted of enzyme extract(300 ,uL),['4C]TRP(1.5 mM;specificactivity0.07 ,uCi/mmol),PALP (25 gM), Na-phosphate(0.1 M; pH 7.6) in a total volume of 400 ,uL.This was incubatedfor 2 h at 25?Cin a scintillation vial. The reactionwas stoppedby adding1 M H3BO3Na2CO3(2 mL; pH 10.0), and T was extractedinto toluene scintillantas previouslydescribed(10). Activity is measuredas nmol T h-1 mL-' of cell fluid. Cell fluid was calculatedfrom the differencebetweenfreshand dried(100?Cfor 1 h) weightsof the seedlings. PIMase and SIMase. The assaymixtureconsistedof enzyme extract(60 ,L), T (for estimatingPIMaseactivity)(1 mM),MT (for estimating SIMase activity) (1 mM), [4C]SAM (0.4 mM; specific activity 0.5 mCi/mmol), and Tris buffer (0.25 M; pH 8.5) in a total volume of 100 ,uL.This was incubatedfor 30 min at 25?Cin a scintillationvial. Activity is measuredas nmol of from SAM incorporatedper h into the tryptamine "4C-methyl alkaloids per mL of cell fluid. The reactionswere stopped by adding 1 M H3BO3-Na2CO3 mL;pH 10.0),and MT (or DMT) (2 was extractedinto toluene scintillant as describedpreviously (10). Extractionand Estimationof Tryptamines TRP. T, MT, and DMT, and TRP wereextractedfrom plant materialas described previously(14). T, MT, and DMT wereseparated estimated and by two-dimensionalTLC (14). This method did not separate TRP from 5-methyoxytryptophan. Accordingly, extractwas the evaporatedto dryness, dissolved in 1 mL of 0.2 M (wrt Na+) citratebuffer,pH 2.2 (12), and estimatedon a JLC 6AH amino acid analyser(JEOLCo. Ltd, Tokyo) using LCR-2 cation exchangeresinon a column (45 cm x 8 mm) at 53?Cat a flow rate of 0.84 mL/min. Threecitratebuffers(12) wereused sequentially for differinglengthsof time, namely(wrtNa+)0.2 M buffer(pH 3.3) for 110 min, 0.2 Mbuffer(pH 4.46) for 50 min, and 0.35 M buffer (pH 5.40) for 130 min. Under these conditions, TRP from 5-methoxytryptophan the otheramino acids separated and in the extractand was eluted at 69 mL after the bufferchange from pH 4.46 to 5.40. The TRP in the proteinsin the seedswas estimatedas follows: 100 mg of pulverizeddry seeds were incubatedfor 18 h at 40?C with pronasesolution (1.0 mL; 5.5 mg/mL in 0.2 M phosphate buffer, pH 7.4). TRP in aliquots (0.1 mL) of the incubation mixturewas estimatedby boilingthe aliquotwith 1.0 mL of van Urk-Salkowski reagent(14) for 10 min, cooling, and readingthe absorbanceat 600 nm. Appropriate controls were included to ensurethat the pronasewas not self-digesting. Reproducibility Results. The concentrationsof intermediof
ates and the activities of enzymes were measured three times.

The trendswere the same in each experiment,but there was a variationby up to half a day of the time at which the plumule emerged through the sand. As the maximal activities of the enzymesand the concentrations intermediates of wererelatedto this event, it was felt it would be more appropriate presentthe to resultsfrom a singleexperimentratherthan as SE means. Total Nitrogen Determination.Forty mg of seedlings were digestedwith 3.0 mL of 18 M H2SO4, 1.5 g of K2SO4, and 0.1 g of HgO at 300?C;NH3 producedwas estimatedby the phenol/ hypochloritemethod (7). Estimationof SAM. Five g of seedlingswere homogenized with TCA (10%;15 mL) and werecentrifuged 2000g, and the at supernatantwas washed with diethyl ether (3 x 15 mL). The extractwas made 15 mmwrt to HCIand then chromatographed as previouslydescribed(5). RESULTS Changesin Tryptophan AlkaloidConcentrations. fluid and Cell weight increasedfrom 8 to 90% of the fresh weight in the first 16 d of seedlinggrowth.Over the 16 d of the experiment,the nitrogencontent of the seedlingsremainedconstantat 105 ? 10 No ,umol/100 seedlings. freeindolecompoundscouldbe detected in the dry seeds, and the amount of TRP in the seed proteins was 5 ,umol/100 seeds. The concentrationof SAM at d 6, the day at which TRP concentrationwas maximal,was 30 ,gM.The earliest time that any of the 3 compounds involved in the synthesisof DMT, namely TRP, T, and MT, could be detected was 3 d afterplanting(TableI and Fig. 1). The concentrationof TRP increasedrapidlyto 1000 ,M in seedlingsat d 6 (Table I). The plumule emerged through the sand between d 6 and 7. the Thereafter, concentrationof TRP decreasedsteadily.Similarly,the concentrations T and MT increasedrapidlyfrom d of 3, the earlieststage at which they could be detected,until d 5, and then they decreased rapidly(Fig. 1). The maximumconcentrations of T and MT were 25 and 53 IAM, respectively.The maximum concentrationof DMT was 650 ,uM at d 8, thereafter decliningquickly. Activities of TDase, PIMase, and SIMase. None of the enzymes, namely TDase, PIMase,and SIMase,could be detected in P. aquatica before the 3rd d after sowing (Table I); they increasedrapidlyuntil just before the seedlingsemerged from the sand (d 6) and then declined until d 14. The maximum activity of TDase was 95 Amol h-'mL-'. The profiles of the changesin concentrationof the two N-methyltransferases were similarand decreasedat a slowerratethan TDase. The maximum activitiesfor PIMase and SIMasewere 1000
Table I. Variation with Time of the Concentration TRPand the of Activitiesof TDase,PIMase,and SIMase in P. aquatica Day 3 4 5 6 7 8 9 10 11 12 13 14 15 16 TRP
MM

TDase 6.77 55.6 94.7 55.9 26.7 17.4 12.0 6.67 2.78 1.16 0.72 0.45 0.27 0.0

PIMase Mmol-'mL-1 222 687 1000 767 606 601 440 381 330 286 217 164 157 150

SIMase 583 1630 2190 1500 1370 1210 982 851 737 639 490 376 343 315

333 444 733 1000 767 826 728 630 533 436 340 267 217 167

ALKALOID SYNTHESIS IN PHALARISAQUATICA

317

50

40-

300

F-20

10

4

8 Day

12

FIG. 1. Variationwith time of T and MT in P. aquaticaseedlings. values for Resultspresented T (0) and MT (a) are fromone experiment; calculatedfromthe numericalsimulationare T (0) and MT (I).

The and 2200 ,mol h-'mL-', respectively. high levels of PIMase and SIMaserelativeto that of TDase could easily convertthe T producedby TDase to DMT and resultedin a low concentration T of the intermediates and MT. Kinetic Analysis. Inhibitiontype nomenclatureand interacfactors to tion factorswereused according Segel(15). Interaction from the are the ratio of Kd-,,from the ternarycomplex to Kd,sS binaryenzyme complex. Kineticconstantsweredeterminedusinginitialvelocityexperiments, and the data were analyzedby a least squarefitting of plots using the intersectingstraightlines to Lineweaver-Burk program Labtech Notebook (LaboratoryTechnologies Corp, Cambridge,MA) run on an IBM PC computer. Data for the Lineweaver-Burk plots were weighted inversely to the Liney(= weaver-Burk I v) value to reflectequalweightingfor each of different the originaldata points. Resultswere not significantly with an unweightedfit, indicatingno bias in the data at high or low reactionvelocity. The mechanismsof TDase and PIMasewere not determined, of and kineticconstantsused steadystate nomenclature Kmand Ki. The binding of TRP to TDase, which was tested at concentrationsof 5 and 25 ,M of PALP,had a smallslope and intercept effect of about equal size giving KP,ALP, 2.5 (,M)). MT and

DMT are competitiveinhibitorsof TDase. K' for MT and DMT for the E-PALPcomplex was high relativeto the concentrations of inhibitorused (1 mM), giving KiMT 5 mM and KiDMT 4 mm. These Ki values were large comparedto KmTRP, consistent with the inabilityof these productanalogsto forman imine with PALP. are Kineticconstantsderivedfrom initialvelocityexperiments listed for PIMase in Table II, for the methylation of MT by SIMasein Table III, and for the methylationof 5MeOMT by SIMasein TableIV. The inhibitionpatternsfor SIMaseare for a rapidequilibrium random bi-bi mechanism with formation of both dead end complexes.The productinhibitionplots intersectnear the axes. The bindingpairs,DMT-MT and SAH-SAM,were good fits to a competitive inhibition mechanism, and, on the assumption that these pairs of ligands bind mutually exclusively,the data to plot werefittedto a Lineweaver-Burk constrained intersecton the 1/v axis. The fits to the graphsfor the DMT-SAMand SAHMT pairswere not constrainedfor the position of intersection. The binding constant for any ligand should be independentof the other ligandsthat are used in the experiment.For example, shouldbe the same whetherit was determinedfor experiKSAM ments with MT or 5MeOMT as the variedligand and with or without any inhibitorspresent.Kinetic constantsvaryby up to a factor of 2 (Table III) indicating that the partiallypurified used in any particular experimentwere not enzyme preparations alwaysidentical.Thus, the magnitudesof the Kdi,svaluescannot be regarded beingmore accuratethan a factorof 2. Inspection as of interactionfactors shows that with the normal number of methyl groupsfor the reactionat the active site of SIMase,i.e. An SAM and MT or 5MeOMT,the ligandsbind independently. extra methyl group, i.e. SAM and DMT, decreasesthe binding of the second ligand by a factor of 3, or, with 5MeODMT, a factorof 2. One less methyl group, i.e. SAH and MT, increases the binding of the second ligand by a factor of 5, or with 5 MeOT, a factorof 2. Model for DMT Synthesis.To determineif the Mathematical
synthesis of T, MT, and DMT could be described in terms of

the enzymes TDase, PIMase, and SIMase, a model for the synthesisof DMT from TRP was constructedfrom the data in Table I and the kinetic constantsin Scheme I. These constants are derivedfrom the kinetic data in Tables II to IV. Velocities for the three enzymes for the numericalsimulationwere calculatedusingthe rateequationforthe mechanism.The mechanism randombi-bi with both dead-end of SIMaseis rapidequilibrium complexes formed. Because of the many similaritiesbetween PIMaseand SIMase,the mechanismfor PIMasewas assumedto

for Table II. KineticConstants TDaseand PIMase Enzyme VariedLigand Inhibitor tion Constant
AM

Interaction

TDase

PALP,TRPb TRPc TRVc MT DMT C C

KmTRP
KmPALP

200
2.5

1.0

KTMT
K?DMT
KMT K,nSAM

5000 4000 17 ? 3

PIMase

T, SAMd Te DMT C

38 ?7
450

1.2 0.4 1.?4

K1DMT

TRP (I100-1000AM) in the C Initial velocities were measuredat eight presence of two concentrationsof PALP (5 and 25 ,M). concentrationsof TRP (40-500 AM) in the presence of two concentrationsof MT or DMT (0 and 1 d Initialvelocitiesweremeasured five concentrations T (0.01-0.075 mM) in the presence four of of at mM). of e Initial velocities were measuredat five concentrations T (0.01of concentrations SAM (5-40 gM). of 0.075 mM)and two concentrations DMT (0 and 1 mM).
C, competitive.

a

b Initialvelocitieswere measured five concentrations of at

318

MACK ET AL. for Table III. KineticConstants SIMase withMT and SAM as Substrates VariedLigand Inhibitor T ipeo Constant
,uM

Plant Physiol. Vol. 88, 1988

Factor

SAM, MTb
MTC

KMT

KSAM

40 ? 6 55 ? 15 120 ? 20 105 ? 10 130 ? 20 35 ? 10 120 ? 40 25 ? 10 45 ? 5 4.3 ? 0.4

1.15 ? 0.20

DMT DMT

C NC

KMT KDMT

SAMd

KSAM KDMT

3.1 ? 0.9 0.18 ? 0.08

MTe
SAM'

SAH
SAH

NC
C

KMT KSAH KSAM KSAH

aC, competitive;NC, noncompetitive. b Initial velocitieswere measuredat five concentrations MT of CInitial velocities were (100-2000 gM) in the presence of four concentrationsof SAM (20-200 ,M). of of measuredat four concentrations MT (100-1000 ,uM)in the presenceof five concentrations DMT (0-1 of at of mM).Concentration SAM = 200 ,uM. d Initialvelocitiesweremeasured 3 concentrations SAM(40of Concentrations MT = 1000AM. e Initial of of in 200 Mm) the presence fiveconcentrations DMT (0- 1 mM). in of velocitieswere measuredat four concentrations MT (100-1000 AM) the presenceof five concentrations at of of SAH(0- 1 mM). Concentration SAM= 200 gM. 'Initialvelocitiesweremeasured fourconcentrations of Concentration MT = 1000 of of SAM (20-200 gM)in the presenceof six concentrations SAH (0-20 AM).
MM.

for Table IV. KineticConstants SIMase with5MeOMTand SAM as Substrates VariedLigand Inhibitor Inhibition Constant
M

Factors

SAM, 5MeOMTb

K5MeOMT

40 ? 10

0.85 ? 0.15

KSAM

90 ? 40 75 ? 10 83 ? 6 66 ? 5 40 ? 10 60 ? 25 7? 4 25 ? 4 2.9 ? 0.3 2.0 ? 0.8 0.45 ? 0.25

5MeOMTC
SAMd 5MeOMTe

5MeODMT 5MeODMT SAH SAH

C NC NC C

K5MeOMT K5MeODMT KSAM K5MeODMT

K5MeOMT
KSAH

SAM'

KSAM KSAH

b Initial velocities were measured at five concentrationsof of four concentrations SAM (20-200 Mm). CInitialvelocities SMeOMT(100-2000 of of were measuredat four concentrations 5MeOMT(100-1000 AM) in the presenceof five concentrations dInitial velocities were measuredat four 5MeODMT (0-1 mM). Concentrationof SAM = 200 AM. concentrationsof SAM (20-200 Mm) in the presence of five concentrationsof SMeODMT (0-1 mM). e Initial velocities were measuredat four concentrationsof Concentrationof SMeOMT = 1000 pM. of of SMeOMT(100-1000 Mm)in the presenceof five concentrations SAH (0-1 mM).Concentration SAM = of 200 Mm. 'Initial velocitieswere measuredat five concentrations SAM (20-200 AM) in the presenceof of of six concentrations SAH (0-20 pM). Concentration SMeOMT= 1000 MM.

aC, competitive; NC, noncompetitive.

Mm)in the presenceof

be the same as the SIMase.For TDase, PALP was assumedto and be saturating the enzyme was treatedas a uni-uni enzyme. Inspection of the experimentallydeterminedconcentrations T of intermediates and MT (Fig. 1) shows that their pool size is small comparedto the amount of DMT producedand that the is rate of change of concentrationof these intermediates small comparedto the flux of the pathway.For all practicalpurposes, all TRP consumed by the pathwayappearsas DMT, and the concentration of the indolethylamineintermediatesis steady occurs on day 4, when the state. The worst case approximation pathwaybeginsto function,when the ratio of the rateof change to of concentration flux is 1:20.The profileof the changesin the of concentrations DMT (Fig. 2, inset)was very similarto that of

TRP (Table I) indicatingthat DMT was being furthermetabolized and was only presentwhen being producedfrom TRP. The behaviorof this systemwas analyzedby numericalmethods usingthe Eulermethod (6), which gives the exact calculated for (Figs. 1 and 2), and, by concentrations all the intermediates assumingthe concentrationof T and MT is small and steady analyticequations,which show how state,derivingapproximate of the parameters the model affectits results. The followinginformationand assumptionswere made about reactantsthat interactwith outside pathwaysand hence are not controlledby the alkaloidpathway: (TableI). 1. TRP: TRP was determinedexperimentally 2. PALP:There are no data for the in vivo concentrationof is PALP;k1,aAeLp low (-2 ltM), and it was assumedthat PALP was alwayssaturating.

ALKALOID SYNTHESIS IN PHALARISAQUATICA
5

319

4
0.6

where, V3 = activityof SIMasesaturated with substrates and VI = activityof TDase saturated with substrates. Prime superscriptsrepresentthe reduced concentration of ligands, i.e. the concentrationof the ligand divided by the Km,
for Ki, or KdisS the enzyme.

The approximationused to derive Equation 1 is reasonable betweendays and 5 and 12, while at day 4 this approximation givesresultslow by a factorof 2 comparedto the exactnumerical E method. For SAM and MT not saturating,Equations 1 and 2 show that SAM has no effect on the flux of the pathway,only 1_ ?0.3 on the concentrationof intermediates. Thus, a decreasein the of concentration SAMleadsto an initialdecreasein methyltransferasevelocity followedby a compensatingincreasein the conof centrationof MT. Since,exceptat d 6, the concentration SAM /s8 16 1is unknown,this propertyof SIMasewas used to test two simple hypothesesabout the time course of SAM concentration.SAM concentrationwhen assumed to be constant led to calculated MT concentrations at which, relativeto concentrations d 6 were 8 16 4 12 too low, the disparityincreasingthe furtherthe age from d 6. Day This indicatedthat the concentrationof SAM used in the simulation is too high at days other than d 6. The concentrationof FIG. 2. Variation with time of DMT in P. aquatica seedlings.Values SAM, when allowed to follow the time curve of TRP, gave calculatedfrom the numericalsimulation;inset, resultspresentedfrom calculatedMT concentrationswhich stayed in step with their one experiment. This latterhypothesis determined concentrations. experimentally in is reasonable, that both TRP and SAM are being producedby 3. Binding of MT and DMT to TDase: KJ0aSfor MT and DMT are large comparedto the concentrationof these ligands the same metabolicmachineryduringmobilizationof the seed storagematerial,and it was used for the resultspresentedhere. and their bindingwas ignoredin the simulation. 4. SAH:No information availableaboutthe concentration was DISCUSSION in vivoof SAH. Since it is a stronginhibitorof the methyltransferases and mechanisms for its removal in other systems are T The concentrations the intermediates and MT are deterof known (8), the concentrationof SAH was assumedto be zero. mined solely by the pathwayand are used as the measure of 5. DMT: DMT is turned over in seedlings of P. aquatica (1), worthof the model. From Equation 1 the errorsin determining and the data presentedhere show that the alkaloidsynthesizing the concentrationof MT are mainly due to three kinetic conpathwayproducesmore DMT than is found in vivo.The model stantswhichhave errorsof a factorof 2 (errors concentrations in of continuallyremovedDMT so that the concentration DMT in or activitiesare only about 10%).In the worst case, where all the model neverexceededthat in vivo.Thus, the enzymesin the three kinetic constants contribute equally and independently, simulation are presentedwith experimentallydeterminedcon- the final error is a factor of 3. Estimates of errors for the centrationsof TRP and DMT. concentrationof T are difficultto determineas, unlike SIMase, 6. SAM:The analyticexpressionfor the concentrationof the PIMaseis not mostly bound to DMT. In this case the expected intermediateMT can be simplifiedfor SIMasewhich is mostly errors for MT are used as an estimate for the error of the bound to DMT giving concentrationof T. The data of Figure 1 show that the ratio for M Flux. DMT' (1) calculated/experimental the concentrationsof T and MT is MT' = ) less than a factor of 3, showingthe pathwayas modelled to be V3 SAM' an acceptablefit to the data. TDaseactivityis the ratelimitingstepin the pathway,resulting in which in bound concentrations the intermediates and MT. RerunT of Flux = activity of TDase = V,TRP'/( 1+TRP') (2) ning the simulationby increasing only the activityof TDase gave
SAM SAM

TRP TRP

A

TDase
200jM RKmP= PALP saturating
TTP

PT

PMase
SM

SIMase
A

DMT

= 4OM KKSAM m = 2OpM
KDMT 450pM =

= K,AM 6Q1M KmT = 4C,jM
KDMT= 6OpM

SAH = OPM SAH = OpM SCHEMEKineticconstantsused in the simulationof the synthesisof DMT in P. aquaticaseedlings. 1.

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PlantPhysiol.Vol. 88, 1988

until TDase reached bound concentrations the intermediates for 0.1 to 0.5 times the activityof SIMase,when the concentrations of T and MT increased withoutlimit. The highestTDase/SIMase ratio found experimentallyis 0.05 on d 5 indicatingthat the pathwayis alwaysmore than a factorof two inside the requirements for a bound solution for the intermediates.Within the time of the simulation,TDase activityvariesover a factorof 350 and TDase/SIMasevaries over a factor of 65. These numbers are largecomparedto the marginof 2 requiredfor maintenance of TDase as the rate limiting step in the pathway, and it is reasonableto suppose that the TDase is rate limiting through selective pressure, rather than coincidence. TRP is close to saturatingat the time when the amount of TDase is highest (aroundday 6) indicating thata changein the pathway produce to unboundconcentrations intermediates of would only come from increasingthe amount of TDase, rather than increasingthe concentrationof TRP. The affinity of DMT for the enzymes is in the order SIMase>PIMase>TDase, decreasing a factorof about 10 for by each enzyme. The very weak binding of DMT to the PALPTDase complex is expected from the chemistryof the TDase reaction,where an imine cannot be formed.The weak (relative to SIMase)binding of DMT to PIMase,when consideredwith the similarchemistryof the two enzymes,indicatesthat PIMase is designed to preclude binding of DMT. DMT only binds strongly to SIMase because it is the enzyme product. Larger amounts of SIMase, relative to the other two enzymes in the pathway,arethus required overcomeinhibitionby DMT. The to enzymesof the DMT-synthesizing pathwaythus arenot designed to use DMT as a feedback-regulating element for the pathway, and the ratio of enzyme activities of the enzymes (in reverse orderof their affinitiesfor DMT) reflectsa requirement low for concentrations the indolethylamine of in intermediates the presence of DMT. The two criteriafor the acceptanceof simulation,the ratio of the calculated to experimentallyfound concentration of the intermediatesand that the concentration of intermediatesis

representation bound,both showthat the model is an acceptable system.We concludethatTDase, of the actualDMT synthesizing PIMase,and SIMaseare the enzymes responsiblefor the in vivo synthesisof DMT.
Acknowledgment-We tions. thank Mr. L. Higginbottom for the tryptophan estima-

LITERATURE CITED 1. BAXTER C, M SLAYTOR 1972 Biosynthesis and turnover of N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in Phalaris tuberosa. Phytochemistry 11: 2767-2773 2. BAXTER C, M SLAYTOR 1972 Partial purification and some properties of tryptophan decarboxylase from Phalaris tuberosa. Phytochemistry 1 1: 27632766 3. COWARD JK, EP SLIsz, FY Wu 1973 Kinetics studies on catechol 0-methyltransferase. Product inhibition and the nature of the catechol binding site. Biochemistry 12: 2291-2297 4. CULVENOR CCJ, R DAL BON, LW SMITH1964 The occurrenice of indolealkylamine alkaloids in Phalaris tuberosa L. and P. arundinacea L. Aust J Chem 17: 1301-1304 5. GLAZER AL PEALE1978 Measurement of S-adenosyl-L-methionine levels RI, by SP Sephadex chromatography. Anal Biochem 91: 516-520 6. GROVE WE 1986 Brief Numerical Methods: An Introduction to Elementary Numerical Methods Emphasizing Iterative Solutions. Prentice Hall, New York, p 98 7. KAPLAN 1965 Urea nitrogen and urinary ammonia. In S Meites, ed, Standard A Methods of Clinical Chemistry, Vol 5. Academic Press, New York, pp 245256 8. KERRSJ 1972 Competing methyltransferase systems. J Biol chem 247: 42484252 9. LEETE ML MINICH1977 Biosynthesis of gramine in Phalarisarundinacea. E, Phytochemistry 16: 149-150 10. MACKJPG, M SLAYTOR 1979 Indolethylamine N-methyltransferases of Phalaris tuberosa, purification and properties. Phytochemistry 18: 1921-1925 11. MARUMP, AW HOvIN, GC MARTEN1979 Inheritance of three groups of indole alkaloids in reed canary grass. Crop Sci 19: 539-544 12. MOORE DH SPACKMAN, S, WH STEIN1958 Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Anal Chem 30: 11851190

13. MULVENA DP, M SLAYTOR 1983 N-Methyltransferase activities in Phalaris aquatica. Phytochemistry 22: 47-48 14. MULVENADP, M SLAYTOR 1982 Separation of tryptophan derivatives in Phalaris aquatica by thin-layer chromatography. J Chromatogr 245: 155157 IH 15. SEGEL 1975 Enzyme Kinetics. Wiley Interscience, New York, pp 274-346

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