a:% Bio$yntheeis of Cyanogenic Glucoeidee in Higher Plants , -* V C OF TNTERMEDUTES IN DHURRIN BlOSYNTHESIS BY A MICROSOMAL SYSTEM FROM SORMuabieIaOU)B (CINN) MOENCH*

Llndberg Meller* and Eric E. Conn ' h m the h-nl of BmhunbW and Biopkv~ic8. Uniumity ofCa!alrfonua, Daum. CaUJbrnco 95616

l%e biwynthetic pathway for the cyanoganic gluco- side, dhurrh, involver the following in t a rmdh tes : LP tyro*, N-hydroxy tpdne , phydroxyphenylacetal- -0, phyhxyphenylacetonitrile, and p h y d m x y -

d e l d M 1 e . N-Hydroxytpsine and phydro ry - nylacetonitrile produced from t ty ros ine by micro- z'

ciomes from needkg~ of Swghwn bifolor are utilized more etYectively a s rubsftaten than (nogenoudy added N-hydrorytyrodne and phydroxyphenylacetonitrile. The minimum values for the dmnueling ratios are 85 for N-hydroxytyrosine and 116 for phydroxyphenyla- catonitrile. On the other hand, phydroxyphenyiacetal- M m e produced internally exchanger reidily with ex- +nously added phydroxyphenylaoetaldoxime. Them wtr indicate that t he bioaynthetic pathway Is cata- IgU)ld.by two multienrcyme complexes o r by two multi- - proteins and explain why the r a t e of the e w eequential reaction starting Prom t ty ros ine is

a n the rates of reaction initiated later in the =with the hewn intermediates N-hydroxyty- w+ne. and phydroxyphenylaeetonitrile. Attempts to mos~link chemically t he l a d enzyme in the pathway, a raluble UDP-glucose glucoeyl-tramsferase, to the mi- myomal system were unsuccessful.

' <

The cyanogenic glucoside dhunin (8-D-glucopyranoeyloxy- S.phydmxymandelonitrile) is rapidly synthesized from L-ty- &he in eeediings of mdan gram Sorghum bicolor (Linn) Mw&. The dry, starch-rich sorghum seed contains no de- *%le dhunin, but the 3-day-old seedling contains 16 pmol

reparation from etiolated hum- catalyze. the conversion of L- tyrohe top

ie. all but the laat ntep in the pathway kt& wmpouMi ir then converted to dhurrin by a

IiY)* ~ ~ ~ ~ ~ Y P E u D P - $ u c ~ ~ #lucoayl transfer= (6). An@,,.6&dscle to unraveling the pathway of dhurrin t

&tX&pyrnsat of p& charpa l l u o utscle mwt therefore be ~sh~aeld'odva)rmmr" mroord.ooe with 18 U.S.C. Seetlon

syntheaia hes been the low amounta of intermediates pnrent in the enzymatic reaction mixtum (3.7). Generally, only ths compound uaed as s u b a t e and the end product, phydrox- ybeddehyde, fonned nonenzymaticdly from p-hydroxy. mandebnitrile (3), are p m n t in quantities d y dehctdble. However, phydroxyphenytacetaldoxims doea accumuhta when sorghum micmmea are prepared in the abwnce of mercaptoethanol and dialyzed under air instead of ni-n (3, 4). p-Hydroxyphenylsoetonitrile accumulaten in d quantities when p-hydroxyphenylacetaldorima is incuhtad with the m i c m m a l system under anaerobic conditioru in the p m n c e of NADPH (4). Accumulation of N-hydroxytyrosine hss only been observed in experiments utiJizing carbon 14- labeled tyrosine as subatrate and large quantities of unhheled N-hydroxytyrosine an a trap (3, 8). In this report. data nm preeented which suggest that the sorghum micmsomm con- stitute a highly organized enzyme system exhibiting catalytic facilitation and thereby providing an efficient mechanism for channeling the flow of carbon from tyrosine into dhurrin. A p r e l i m i i report of theee fin- has a l w d y appeared (9).

EXPERIMENTAL PROCEDURES

Chemwala-Bu(methyl)wberLni&te WM nyntheeLed Prom sub. emnitrile (10). All other chemicab were ~,yntheitzed or purchwed M denmibed earlier (3, 11, 12). L-[U-''C]TYIO.~~~P (specific activity, C(O mCi/mmol) and ~-[rurcl-2,3,6.6-~H]tymsine (specific activity, 90 Ci/ mmol) were pumhusd bom New Enghd Nuclear, Bwton, MA.

Chcmunl Syntheara of Radwactiw Inkmudiatca-Chsmicrl synthean of uniformly "C-labeled N-hydrorytyrwine, p-hydmry- phenylacetaldoxime. andp-hydmxvphenylacetonitrile WM wrhd out M &tier danibed with L-[U-"C~& an the starti* m a w (7). The N-hydmxytyrorine wrs pwifmd by ion exchange chmmato(l. raphy (7). pHydroxyphenylacetaldoxime and p-hydroxyphmykoe. tonitrile ware puritbd by preparative thin layer chmnmtngmphy (7) and were radiochemically pure when loalyled by the GLC/GPCf procedure (11) and by ranning thin layer chmmroguns with a radiochmmatogram wanner (Packud model 7201).

Analyare of Radwactiw InknnedrotrtRaaction mixturn which did not contain N-hydroxytyrooine were analyzed by thin layer chro- matcgraphy (3,ll). The TLC platm (Bakeflex IB-F flexible Ih.et.) wen pmtmaked with unlabeled standuda to allow &*tion under ultraviolet light lftcr chmmtognphy. Aliquot. bwn biayn- thetic m h n mixtursll were streaked d i l y on the plabr. '&& d r c d in immediate inactivation of t h microromal anzymc ayatem M judged by Be linenrity of the naction with Lime (3). The TU: platg were developed in bewne/ethyl acetate (I:?, v/v) and t b radioactivity in mpanted compoun& was detarmrned by at* appropriate araaa into counting viala. The Rp valuea obtljllbd wern: tyrmiw, 0.00; phydroxybmzoic acid, 0.06; p h y d m ~ y p h e n ~ w . doxime, 0.17; phydmxybeiddehyde, 0.W ud p-hydmr-y~~ mtonihile. 0.42.

-- - I The abbnviatiow rved m: CLC/CPC, gla-liquid c h m ~ t o ~ . .

phy/ys proportiod wntins, Tncine, N-[TrL(hydroxm. fl)methylJgiycine; lldodms,p.h~droa~~hen~1.ot.dd~; nitrib,p hydroxyphmnylacebnierile; aldehyde, p-hydroxybenuld.hyde.

Cyanogenic Gluc

Reaction mixtures containing N.hydroxytyrcuine. which accumu- latea only under a m i d conditions (3). were analvzed with a nas chromatograph ( ~ l k ) coupled to a gas proportionai counter (GPE), as earlier described (11). Thii was necesRary because N-hydroxyty- roehe oxidatively decarboxylates, forming p-hydroxyphenylacetal- doxime, when a n d y d by T W (7). Aliquotsfrom enzymatic reactions were lyophilized to dryness and silylated by refluxing for 16 min at 90'C with an excess of N,O-bis.(trimethylsilyl).trifluoroecetamide in acetonitrile. An unlabeled standard mixture of trimethyMyl-deriva- tized intermediates was also added. Aliouotn were iniected into the GLC/GPC equipped with a SP-2250 coiumn. The temperature pro- gram consisted of an initial period of 6 min at lSh°C, a temperature riae of 3O0C/min, and a final period of 12 min at 17S°C. The area of each labeled peak obtained was intearated electronicallv and the wak was identified by retention time and superimpusition kith the mass wak of the authentic standard. M m and radioactivitv tracines bbtained wing the GLC/GPC procedure have been preG- oudv (3).

he preeence of dhurrin in microeumnl reaction mixtures was a h analyzed by uw of the GLC/GPC procedure after trimethylsilylation of the sample. Since the molecular weight of trimethylailylated dhur- rin is large compared to those of the derivatized intermediates, the regular temperature program was expanded to include an additional temperature rise of BO°C/min and a final period of 12 min at 255°C. with thin program, dhurrin was elut.ed ifter fi.5 min at thia final temperature.

delerminatiun of Sperific Activity--The commercial wmples of L-[U-"C] and 1,-[ri~2,3,5,6-~H]tymsine were diluted to suitable s w - eific activities by addition uf known amounts of unlabeled I.-tyronine. The diluted material was then recrystallized froin ethyl alcohol-HAD and its specific activity was determined by liquid scintillation counting of a weighed aliquot. The specific activity of intermediates synthesized from L-[U-"C)tyronine was obtained from t,he npecific activity of the L-fIJ-"Cltvrosine used bv correctinn for the loen of 1 or 2 carbon ahmn d"hng the synth&is of p-h~droxyphenylacet~ldoxin~e or p- hydroxyphenylacetonilrile, andp-hydroxyben7~ldehyde. rerrpectively.

Measurement of Radioac~lroity-Redioactive samples (crystalline material. aaueou samolex, or radioactive nrmts cut out from T1.C plates) weremeasured after the addition of a'tatai of 1 ml of HIO and 10 ml of a counting wlution compoaed af 0.5 liter of Triton X-IIWJ, I titer of toluene. 7.50 g of 2.5-diphenyloxawle and 0.30 p of 1.4-big2- (5-phenylozaaolyl)]benzene. All samples were kzpt in the dark for at least 2 days before counting in a Searle Mark I1 scintillation counter usinn the discriminator settine8 of I'ronram OH desiened fur samr>lea containing both ''H and "C. b d e r these conditions, there wa; no spillover of 'H into the "C channel nnd the cpn1 in the "C channel wan therefore converted directly to dpm of '% by use of the external atandard ratio. Correction for upillover of "C into the 'H channel wax made using e calibration c w e obtained by counting a aeries of samplss containing only "C. After subtraction of the "C xpillover, the remaining cpm in the 'H channel were converted to dpm of "H by meana o f the external standard ratio. In enzymatic experiment.% the % and "C dpm values observed in the diflerent metabolites were converted to nanomoles of metabolite by w e of the known specific activity of the ~uhstmte employed. In thii convesion, correction was made for the loss of 1 or 2 crubon atom8 when productn were formed from uniformly "C-labeled tyrosine.

Seed Malerid and Mierosomal Preparatiom-Seed of S. hicolor (Linn) Moench were obtained fmm Northmp, King & Co. Lubbock. TX. The hybrid Sordan 70 was uaed in the investigation of the effect of dialysis time on the accumulation of intermediatm (3). The hybrid Uedland x Greenleaf was used in all other experiments. ~ ic ro&mal preparations were obtained as earlier described (31, unless otherwise indicated.

Experiment8 to Detect Enzyme.bound Inlermcdrateti-The pro. duction of bound intermediatea was analyzed with the following "C- labeled substrates: L-tyrosine (specific activity. 2.50 mCi/mmol), N- hydruxytyme~ne (spmfic aclinty. 2 50 mCl/mmolJ, p-hydrox.yphen. ylacetaldonme (hpecfic actmty. 222 mC11mmnl~. and a-hvdroxv- pheny~acetonitril~ (specific activity. 2.22 mCi/mmol). Eaih kact&n mixture contained 0.2 nmol d "C-labeled wbatrnte. 0.3 umol of NADP+, 3 pmol of glucase-6-P, 3 ID of glucose-6-P dehydrbgenaae. 3.0 nu of miwsormrl ~mte in . and W umol of Tricine (DH 8.0) in a total colume of 810 yl: Aliquotn (150 pl) were removed'ht 5, 10, 30, and 60 min and pipetted into m a l l plastic centrihge tubes containing either 90 pJ of 1 W wichloroacetic or 200 pi of ethyl alcohol. h i p itatad protein was imhted by centrihption, and after five repetitive w a s h i with a09h trkhlomcetic acid or 6(rX ethyl alcohol m p w

tively, the protein precipitate was resuspended in 1 ml of 1 N HCI and counted in a liquid scintillation counter. In a second aeries of experi- ments, with reaction mixtures ident id to thoee d w r i h d above and 3Chmin incubation time, the miemaomal protein was recovered from the reaction mixture8 by ultracentrifugation (100,000 x g for 30 min), washed. and counted as demibed above.

The b i b l e production of activated intermedintea (i.e. coenzyme A derivative8 or nhoanhates) was invurtkated with '%-labeled tvm- sine as substrate.i.he;eaction mixtures were asdewxibed abbve, except that, in one experiment, 1.2 pmol of unlabeled N-hydroxyty- roaine were added as a trap. After 30-min incubation, the reaction mixture8 were transferred to an ice bath and an eaual volume of 12 N HCI was added. After standing for 3 days at r~om'tem~erature. the reaction mixtures were Ivoohilized and their content of labeled inter- mediates was assayed l;y 'the GLC/GPC procedure and compared with the content of unhydralyzed reaction mixturan. N-Hydroxyty- rosine is stable under these conditions (7) and therefore could be memured. p-HydroxyphenylacetaIdoxime, however, undergoes some decomposition in acid and was therefure not quantitated. Alkaline hydrolysis could not heemployed an N-hydruxytyrosme would decom- pose into other intermediaten and l~roduce amhiguwus results (7).

Experrm~nlv In~,olomn~ S~multaneous Incubahon of "H- and "C. labeled Subrtra1e~-The reaction mixtures contained 0.2 pmol of NAI)P'. 2 umol of rlucorw-6-1'. 4 I11 of nlucose-6.P dehvdrorenasc. . . . .. 0.60 mg of niicrosomal protein, 15 pmol of phosphate buffer (pH 7.2). and 0.160 pn~ol of each radioactively labeled subwtrate (Table 1) in a total volume of 202.5 pI. The specific activitieu of the labeled sub. straten were: L-I.'Hltyrosine. 7.36 mCi/mmol; ('4Cb-hydmxypheny. lacetaldoxime, 6.39 mCi/mmol; ["Clp-hydroxyphenylacetonitrile, 2.49 mCi/mmol. In one exl~riment, the dctergent. Tritc~n X-100, wau added to a final concentration of 0.05%. Aliquota (:M p1) were taken at T - 4. 10. 16. 20.00. and 60 nun and analwed bv the TI,C ~rocedure. . . . . "

Experimentti Intwloin# .Sequential Inrubabr,n of 'H- and "C- labeled Substralea-The reaction mixtures contained 0.3 w o l of NADP', 3.0 pnml of glucc,ar-fi-1'. 4 II1 of glucose-6-1' dehydrogenaae, 0.45 mg of microwmal protein. 25 pmol of phosphate buffer (pH 7.2). and 0.160 pmol of a radioartivsly labeled substrate in a total volume of 162.5 pl. Aliquots (15pl) ware taken at T - 2.4,6, and 8 min. At T - 8 min (Fig. 2). 0.lW pmol of a differently labeled substrate were added 140 ul) and a second se r i r~ of aliuuotx 125 ul) were made at T - 10, 12, lb;. 18, and 20 min. The specil$ activities of the substrate8 used were as indicated above. All aliuoota were analvzed hv the TLC . .. procedure.

fi:xperrmentti h~ Determine the E'xrhanpe between Internally Pm- durrd and Externally Arldcd N-Hydroxytymsine-Reactions were carried out in I-ml ampul~w. The reaction mixtures used contained 0.2 pmol of NAIIP', 2 pmol of glucose-6-P. 4 IU of glucose-6-P dehydrogenme, 2.0 mg of microeumal protein, 30 pmol of Tricine buffer (pH 8.0). and 0.16Opmol of each subatrate (Table 11) in a total volun~e of :M pl. The specific activities of the substrates were: ["Cltyn~sine, 5.00 mCi/mmol; N-hydr~~xg["C]tyrosine. 3.37 mCi/ mmol. After 20-min incubation, the ampules were immerned in liquid nitrogen to stop any enzymatic reaction. Their cuntentn were then lyophiiized to dryness, silylated, and analyzed by the CLC/CPC procedure.

Effect of Dialysis Time on the Accumulation of Intermediotcs- Micra9omes from etiolated sorghum seedlings (S. bicolor (Linn) Moench C.V. Sordan 70) were prepared without mercaptoethanol as earlier described (3) and aliquots (1 ml) were dialyzed against 20 mm Tricine buffer (pH 8.0) for different time periods (Fig. 3). At each time period, the mic rm en were assayed in reaction mixtures con- taining 0.3 p o l of N A D a , 3.0 pmol of glucose-6-P, 4 IU of glucose- 6-P dehydrogenase, 2.0 mg of microsomal protein, 30 pmol of Tricine (pH 8.0). 0.060 pmol of L-["C]tyrosine (specif~c activity. 16.71 mCi/ mmoll in a total volume of 13.5 yl. The incubation time used was 16 min and aliquots were analyzed by combined use of the T v gn4 GLC/GPC proceduran (3,11). I _ ,I

Experiment8 to Cro88.link the IIDP-Glucuw~ ~lucoayltra~&&~ to the Micrommal Comulex by Use of an Imidak-Etid.ted sorghum lleedlings (10 g) were homogenimd in a freshly prepared medium (20 d) contaiPina 3 mmol of triethanolamine. 7 mmol of NaCI, 0.3 lunol of mer&ptoethsnol, 0.2 mmol of bh(methy1)- . nuberimidate and I g of polyvinylpolypyrrulidone at pH 8.2. M i c v mmes were obtainod by differential centrifugation, as earlier de. scribed (3). In a contml experiment, bii(rwthyl)wberimidrte *as omitted from the homogenhtion buffer. The -tic sctivlty d*l the micmeod prepuationn ms uaayad in mction mhttureB mul- paced as deaaibed in the preceding section except Ulnt 2.0 p o l of

Cyanogenic Glucoside Bios.vnthesis 305 1

UDP-glum were added to each resctiun mixture. The reaction The low amounts of intermediates prewnt in the mirro. mixtures were incubated for 30 min and then -lysed by the somal reaction mixtures (Table 1) and the earlier relwrted CGL/GPC procedure as modified for dhurrin detection. dificulties in trapping both N-hydrox.yt.yrosine (3) and p-

hydroxyphenylacetonitrile (4) are understandable if the reac- RESULTS tionn of the bitmynthetic sequence (Fig. 1) take place in 11

Analysis of Reaction Mixtures for Intennedintex-When highly organized enzyme system w) that intermediates pro- sorghum microsomes are incubated with radioactively labeled duced internally are preferentially utilized by the riext ensyrnr L-tyrosine orp-hydmxyphenylacetaldoxime (aldoxime) in the in the sequence. Such intermediates would not diwociate presence of NADPH, p-hydroxybenzaldehyde iR the major readily from the surface of the enzymes involved and therefore product formed (Table I, Experiments 1 and 3). Whiie the would not exchange readily with intermediates added from aldoxime andp-hydroxyphenylacetonitrile (nitrile) are entab- outside. T h i ~ powibility was investigated in experiments uti- liahed intermediates in the conversion of L-tyrosine to p- lizing two substrates labeled with different radicri)topes. hydmxy-(S)-mandelonitrile, they are present in concentra- Experiments lltiliring Simultnri~oux Addition of Tuv) I)i( tions 1 order of magnitude smaller. The low amounts of ferently Labeled Substrates-In one t.ype of experiment, two intermediates observed could be the result of such interme- substrates labeled with different radioisotopes (.'H and "C) diates being enzyme-bound during biosynthesis. This pomi- were administered simultaneously to the sorghum microxomal bility was ruled out by reisdation of the microsomal protein system in the presence of NA1)PH. The amount of each after incubation with radioactively labeled substrates in the suhntrate used was sufficient to mturate the enzyme system presence of NADPH. The pn~tein w a ~ isolated by precipita- for incuhation periods up to 30 min. Aliquotn taken at different tion with either trichloroacetic acid or ethanol or wan re- time periods were analyzed for labeled intermediates by thin covered by ultracentrifugation of the incubation mixtures. In layer chromatography and the content of 'H and "C in each no case did the isolated protein contain radioactivity. Acid intermediate was determined by liquid ~cintillation counting. hydrolysis of the reaction mixtures also did not produce From t,he known specific activities of the 'H- and "C-laheled additional amounts of free intermediates. The results thus substrates emplo,ved, the nanomoles of intermediates formed indicate that none of the interniediates are covalently bound both from t,he "H- and the "C-labeled precursor were calru- to enzymes and alwo suggest the abwnce of quantitatively lated separately. Data obtained after 20 min of incuhation nrr significant amounts of activated intern~ediates such as coen- preuented in Table I (Experiments 5 and 61. Qualitatively zyme A or phosphate esters. The Ibrmation of such activated similar data were obtained at 4. 10, 16. :$(I, and MI min of intermediatm would presumably alfio have been indicated by incuhation. When f 'H]t,yrosinr and ["CJp-hydroxyphenylH- specific cofactor requirements of the microwmal system. cetaldoximr were used sinlu~taneoudv a8 suh~trater; ('l'ahlc 1.

TAHI.Y. I The melahlrnrn of rrrdroarlil~ely lahebd xubvlrntes tn mtr,roxomnl rmc.lmrt nrtxlurrs trt 1huprrs~nc.r of NAI)JIH

Saturating amount8 of each auhntrate (I(% nmol) were uned in all exprrin~c*nLr. 1)nI~ prc.rmt-cl are for an rnrubali~~n tir111. 111 4) min. For further exwrimental detaile. we "Exverimental l'rouedures."

1 ['HJTyrofiine 1 28 .'i.ti 2 ['HJTyrofiine + 0.05'!+ Tri- 1:W IO.li

ton X-100 3 ["C]Aldoxime 4 [''CINitrile 6 [,'H]Tyroe.ine + ["Claldox- 14.1 10.9

une 6 [%]Tyrosine + ["C]nitrile 142 2.5 -- . - .o(JcH2-rH

NOH

8 qlucosr p-hydrosy- p-hydroxyphenyl.

dhurrin rnonfhbn~trile ocelon~lr~le

FIG. 1. Bionynthetic pathway for the cyanogenic glucoside dhurrin.

3052 Cyanogenic Gluco 'side Biosynthesis

Experiment 5), the molar :'H/I4C ratio of the aldoxime imlated from the reaction mixture after 20 min of incubation was 0.133. The molar 3H/14C ratios for p-hydroxyphenylacetoni- trile and for p-hydroxybenzaldehyde isolated after the same time period were slightly lower, namely 0.098 and 0.091, respectively. Thus, the aldoxime added externally competes effectively with aldoxime formed in xitu from tyrosine during conversion of the amino acid intop-hydroxyphenylacetonitrile and p-hydroxybenzaldehyde and there is no channeling.

Further analysis of Table 1 diwloses that a total of 69 nmol of p-hydroxybenzaldehyde were produced from ["Hltyrosine and rl'C]aldoxime incubated simultaneously with the sorghum microsomes (Table 1, Experiment 5). When the two nubstrates were administered separately to the microsomal sywtem (Table 1, Experiments 1 and 3), a total of 117 nmol of aldehyde were formed. The main reason for the increased production of p-hydroxyhenzaldehyde when the substrate^ were administered separately is t,hat eaturating amount8 of each uubst.rate were used. Moreover, the ["Clp-hydroxyphen- ylacetaldoxime used as substrate in Experiment 5 acted as a trap for ["Hlp-hydroxyphenylacetaldoxime formed from ["Hltyrosine in that experiment. A hetter measure of meta- bolic activity in these experiments iu the amount of substrate which is utilized. When the two substrates were administered separately (Table I, Experiments 1 and 3), a total of 136 nmol were metabolized. In Experiment 5, this value was 95 nmol. From the known composition of the reaction mixture in this experiment, it can be calculated that this lower value is obtained because a saturating concentration of aldoxime in- hibits tyrosine metabolism 48% while a aaturating concentra- tion of tyrosine inhibits aldoxime metabolism only 25%.

A similar experiment was performed in which ["Hltyrosine and [l'C]p-hydrtuyphenylacetonitrile were administered si- multaneously to the microsomal s,ptem (Table I, Experiment 6). The molar "H/"C ratio ofp-hydrox.yphenylacetonitrile and p-hydroxybenzaldehyde reisolated from the reaction mixture after 20 min of incubation was 0.012 and 1.32, respectively. These figures indicate that p-hydroxyphenylacetonitrile added externally is not able to compete eficiently with p- hydroxyphenylacetonitrile produced in situ from tyrosine dur- ing conversion of the latter into p-hydroxybenzaldehyde by the microsomal enzyme nystem. A channeling ratio of 1.32: 0.012 or 112 may be calculated, and this means that not more than 1 molecule of p-hydroxyphenylacetonitrile ("H-labeled) exchanges with the pool of externally added nitrile ('%-la- beled) when 112 molecules of ["Hltyrosine are converted to ["Hlp-hydroxybenzaldehyde via [:'HIp-hydroxyphenylaceton- itrile.

When saturating concentrations of tyrosine and p-hydrox- yphenylacetonitrile are administered simultaneously to the microsomal system, a total of 36 nmol are metabolized (Table I, Experiment 6). When the two ~ubstrates are administered separately. a total of 51 nmol are metabolized (Table I, Ex- periments 1 and 4). Fmm the composition of the reaction mixtures (Table I, Experiments 1, 4, and 6), it again can be calculated that the saturating concentration of p-hydmxy- phenylacetonitrile inhibited tyrosine metabolism 4646 while the saturating concentration of tyronine inhibited p-hydroxy- phenylacetonitrile metabolism only 10%.

Experiments Utilizing Sequential Addition of Two Differ- ently Labeled Substrates-In a second type of experiment, the microsomal system was4nitially incubated with only one substrate. Then, after a period of metabolism, the second and differently labeled substrate was added. Aliquota were taken during incubation to calculate the rate of conversion of each substrate into p-hydroxybenzaldehyde. Again, the concentra- tiom of the subtratea wed were at saturation even after

metabolism and dilution of the reaction mixture uponaddition of the second mbstrate. Preliminary experiments with each substrate incubated separately with the microsomal system a h 8howed that the production of p-hydroxybenzaldehyde was linear with time.

When ["Hltyrosine was used as the first substrate, the rate of [:'HIP-hydro~ybenzaldeh~de production was 141 nmol/mg of protein/h (Fig. 2 4 . Upon addition of ["Clp-hydroxyphen- ylacetaldoxime at 8 min, the total rate of p-hydroxybenzal- dehyde production from both compounds increased to 201 ~ n o l / m g of protein/h. However, most of the phydroxyben- zaldehyde now formed (191 nmol/mg of protein/h) was de- rived from the ["C]p-hydroxyphenylacetaldoxime added at 8 min, and the total rate ofp-hydroxybenzaldehyde production after addition of both substrates was still less than half the rate obtained whenp-hydroxyphenylacetaldoxime was admin- istered initially as a single sobstrate (452 nmol/mg of protein/ h) (Fig. 2 8 ) . When, under the latter conditions, a saturating amount of [''Hltyrosine was added at 8 min (Fig. 2 8 ) , the total rate of p-hydroxybenzaldehyde production decreased only slightly to 414 nmol/mg of protein/h. Hadiochemical analysis of the product now formed disclosed that 402 nmol ofp-hydroxybenzaldehyde were derived fromp-hydroxyphen- ylacetaldoxime while only 12 nmol originated from tyrosine. These two experiments (Fig. 2, A and 8) thus show that nearly all of the p-hydroxybenzaldehyde produced derives from p-hydroxyphenylacetaldoxime when tyrosine and p-hy- droxyphenylacetaldoxime are both present. This is the result expected if the ["HJp-hydroxyphenylacetaldoxime produced in situ from ["Hltyrosine exchanges readily with the large quantity of externally added '"-labeled p-hydroxyphenyla- cetaldoxime.

Similar experiments mere also carried out with ["Hltyrosine and ["C]p-hydmxyphenylacetonitrile as substrates. With ["Hltyrosine as the fwst substrate and the later addition of a saturating amount of [l'C]p-hydroxyphenylaceto~trile, a de- crease in total p-hydroxybenzaldehyde production from 142 to 106 nrnol/mg of protein/h occurred (Fig. 2C). This addition of [14C]p-hydroxyphenylacetonitrile only decreased the pro- duction of p-hydroxybenzaldehyde from [3H]tyrosine to 78 nmol/mg of protein/h. When ["CJp-hydroxyphenylacetoni- trile was used as the fwst substrate, the later addition of ["Hltyrosine at 8 min resulted in a total production of 44 nmol of ['Hlp-hydroxybenzaldehyde/mg of protein/h (Fig. 2 0 ) . However, the production of p-hydroxybenzaldehyde from [l'C]p-hydroxyphenylacetonitrile decreased dramatically at that time from 99 to 10 nmol/mg of protein/h. These results thus again indicate that p-hydroxyphenylaceto~trile pro- duced in situ does not exchange to any uignificant extent with the pool of p-hydrc~xyphenylacetonitrile added externally.

Microsomal Metabolism of N-Hydroxytyrosine-N-Hy- droxytyrosine cannot be analyzed and quantitated by the TLC procedure used a b o ~ (7). Therefore, a gas-liquid chmmato- graphic procedure was employed which involved the separa- tion of trimethylsilyl-derivatized intermediates and analysis of their "C content by combustion to ["CICOZ and continuous meaauremenb in a gas proportional counter (11). Since the method could not be used for 'H counting, this study of N- hydmxytyronine metabolism involved a series of experiments in which the lnicrosomal system was incubated separately or simultaneously with tyrosine and N-hydroxytyrosine alter- nately labeled with I'C (Table II). In thh way, the metabolimn of the two compounds could be detmmined as well as the intluence of the other substrate on the rate of metabolism determined

Table II ( h p e h e n t a 1 and 2) nhows that N-hydroxyty- mine metabolism is inhibited 66% by the addition of tyrohe.

Cyanogenic Glucoside Biosynlhesis

Fa; 2. Hate of phydroxyhenral- dehyde production from differently labeled radioactive compounds. l'hr rxperin~entr wen. slant4 w ~ t h the uinyle rumpound" indicated by the nrvotc, 111 rent tinre. The necunil com(r~unrl nddrd i r ~ each of the four cxperln~ellln IA. B. ('. and 0) is ir~dlcnled hy an arrort.111 H For further rxl~erimrn~r~l rlrl~,ils. RI*~. "b;xperimental 1'rocnlurt.x."

TIME, rnin

TAHI.E I1 Mrtnboli*m of N-hydmrytymsinr and eroninr ly the m~rroxornul x.yntem

Data vresented are for an incubation time of 20 mill. For exwrimrnLai drtailn. see "Exoerimunlul I'roredurch"

~- - - - ~

IN) nmol nmol 1 N-Hydroxytyrnnine None 41; 36 (1 29 2 N-Hydroxytyrusine Tyroaine I6 9 0 10 3 Tyruvine None 98 7U 0 (i l 4 Tyrosine N-Hydroxytymsinr 81 71 ,'{,I; 57 - ~ -- -. -- -- - -- - - - .

However, the presence of N-hydrox.ytyrosine inhibited the metabolism of tyronine only 7% (Table 11, Experiment8 3 and 4). When 91 nmol of [L'C]tyronine were metabolized in the presence of a saturating, unlabeled N-hydroxytyrosine trap, only 3.6 nmol were trapped as N-hydroxy("C]tymaine (Table 11, Experiment 4). This correspond8 to a channeling ratio of 25. However, 3.6 nmol is the minimum amount of N-hydrox- ytyrosine produced in situ which exchanged with the exter- nally added pool because approximately 108 of the externally added N-hydroxytyrosine pool was also metabolized during the incubation (Table 11, Experiment 2). A total of 4.0 nmol of N-hydroxy["C]tyrouine, therefore, had equilibrated with the externally added N-hydroxytyroaine pool during the ex- periment. Thus, aa with p-hydroxyphenylacetonitrile, inter- nally produced N-hydroxytyrouine exchanges only to a very limited extent with an externally added pool of N-hydroxy- tyrosine. The addition of a saturating amount of L-tyrosine inhibited N-hydroxytymsine metabolism by 65% (Table 11, Experiments 1 and 2) and p-hydroxyphenylecetonitrile me- t a b o h by 10% (Table I, Experiments 4 and 6). Even with this inhibition taken into consideration, high channeling of N- hydmxytyroaine and p-hydroxyphenylscetonitrile are ob- served. Contrariwkie, phydroxyphenylacetaldoxime readily e x c h g w with an externally added pool. .

Effect of L)~alyfiin-It has previously been reported that when microsomni are prepared in the absence ol' mercapto- ethanol and dialyzed for 18 h. the mejor product obtalnsd upon metabolism of tyro~ine kp-hydroxyphenylacetaldoxin~c instead of p-hydroxybenzaldehyde (2, 3, 7). The effect of dialyuk time on thiu result wax investigated more carefully (Fig. 3) in the present study. With the experimental conditions used, dmoxt complete utilization of the tyrouine occurred. The enzymatic reaction is therefore not linear with time. Furthermore, additional convernion of the acrumulated p- hydroxybenzaldehyde into p-hydroxybmzoic acid wnfi alno observed. It was found that p-hydroxyphenylacetaldoxime accumulated when the period of dialysis w a ~ longer than 14 h. Microsomal preparations which accumulatedphydrox.~hen- ylacetaldoxime aLw did not metabolize p-hydroxypheny1ac:e- tonitrile.' Mercaptoethanol is an inhibitor of Rome proteases (13). The accelerated inactivation of the enzyme syst.ern with time in the ah~ence of mercaptoethanol could thus be cawed by proteolytic degradation. A second explanation could be the facilitated oxidation of an ewntial sulfhydryl group in the absence of mercaptoethanol. Preliminary experiments with different detergents which solubiliid the micrommal ~ystem

'B. L. Meller, unpublished results

3064 Cyanogenic Gluco side Biosynthesis

Dia lys is Ttme

FIG. 3. Effect of dialysis time on the accumulation of inter- mediatea formed from L-tyroslne. Fur further experimental dr- tails, see "Experimental Procedurert."

resulted in complete low of enzymatic activity, Treatment with a lower concentration of detergent (Table I, Experiment 2), which did not soluhili7x the microsomes, re~ulted in in- creased formation of aldoxime.

Fmm the data presented above, it appears that the particle- hound enzyme system catalyzing the earlier enzymatic steps in the hiosynthesin of dhurrin is highly organized. It is there- fore surprising that the very laut enzyme in the pathway, the UDP-glucose glucosyltransferane which converb p-hydroxy- mandelonitrile into dhurrin (Fig. I), is a soluble enzyme (6). We therefore questioned whether the glucosyltransferane is actually a part of the microsomal complex which dlasociates during preparation of the microaomes. To ~ tudy this question, the initial homogenization of the seedlings was carried out in triethanolamine buffer containing a bifunctional irnidate, bis(methyl)suberimidate. The imidate will covalently link neighboring proteins if these have a free lyuine c-amino group in suitable position, and might therefore possibly bind the glucosyltransferaee to the micro~omal complex. If this oc- curred, and if the enzymes were not inactivated by the treat- ment, dhurrin inatead of p-hydroxyhenzaldehyde should be the end produd obtained. When the metabolic activity of such craaa-linked microsornal preparations was examined, it was found that tyroaine was metabolized into p-hydroxyben- ddehyde at the same rate as with untreated microsomes and that no dhurrin wan formed.

DISCUSSION

A multienzyme complex can be defined as an aggregate of different, fundionally related enzymes bound together by noncovalent forces into a highly organized structure (14). A multifunctional enzyme consists of a single polypeptide chain with multiple catalytic functions (16). In both systems, active centers catalyzing sequential reactions can form a composite active site which allows the intermediates to channel (16). A well characterized example of a channeled system is the soluble "aromatic complex" of Neumepora crassa, which catalyses five consecutive reactions in the shikimic acid path- way leading to the bimyntheaia of aromatic amino acids (17). Thin system was originally thought to be a multienzyme complex containing five different polypeptides (18). Genetic analysis (19,20) and improved isolation technique8 (21) have now shown that the aromatic complex is a single polypeptide

with five active sites (21). The soluble kaurene eynthetase of higher plants (22) is another example of a bifunctional enzyme system capable of metabolic channeling (23). Due to the limitation of experimental techniques, few equivalent studies on membrane-hound enzyme systems are available (14). How- ever, phenylalanine ammonia lyase and two cinnamic hydrox- ylases, key enzymes in the biosyntheriis of l i i n s , flavonoids, phenolic acids, coumarins, and stilbenes have recently been reported an wembled consecutive enzymes on micmomal and chloroplast membranes of plants (24,25).

Several lines of evidence suggest that the enz.ymes catalyz- ing the biosynthesis of dhumn constitute an organized mem- brane-bound system capable of efficient metabolic channeling. First, the sorghum membrane preparation catalyzes a multi- step vonversion (2). Second, the formation of each individual intermediate in the reaction sequence can be shown but the experimental conditions required to demonstrate their for- mation varies (3. 4). Third, kinetic analysis shows that the particles preferentially utilixe t.yrosine and p-hydroxyphen- ylaretaldoxime instead of N-hydroxyt,yrosine andp-hydn)xy- phenylacetonitrile ( 3 , 4 ) . (The low r a b of nitrile utilization is esperinlly surprising since its hydroxylation to formp-hydrox- yrnandelonitrile is proposed as the next to last step in the sequence (Fig. 1)) . That the membrane system channels the flow of carbon from tyrosine into p-hydroxymandelonitrik is shown by the channeling ratios of 26 for N-hydroxytyrosine and 115 for p-hydroxyphenylacetonitrile obtained at satura- ting substrate concentrations. Similarly, when a saturating concentration of N-hydroxytyrofiine or p-hydmx,vphenylace- tonitrile is added to a membrane preparation actively metab- oliainy tyrosine, the p-hydroxymandelonitrilr subsequently produced still, to a significant extent, is derived from tyrosine. These data thuu demonxtrate the channeling of the interme- dia te~ N-hydroxytyrosine andp-hydroxyphenylacetonitrile in dhurrin biosynthesis.

By contrast, p-hydroxyphenylacetaldoxime produced inter- nally from L-tyrosine exchanges freely with exogenous aldox- ime at saturating substrate concentrations and there is no preferential utilization of either form. Moreover, when p-hy. droxyphenylacetaldoxime is added to a memhrane prepara- tion actively metabolizing tyrosine, thep-hydroxymandeloni- trile subsequently produced is derived mainly from the aldox- ime (Fig. 2A). These results indicate that the sequence of reactions converting tymsine intop-hydroxymandelo~trile is catalyzed by two bifunctional system, the fvst channeling the flow of tymsine intop-hydroxyphenylacetaldoxime via N- hydroxytyrosine and the second channeling the flow of p- hydroxyphenylacetaldoxime intop-hydroxyrnandelonitrile via p-hydroxyphenylacetonitrile. This hypothesis would agree well with the observed accumulation ofp-hydroxyphenylace- taldoxirne after prolonged dialysis (Fig. 3) or after treatment with detergents (Table I,,Experiment 2), indicating the specific inactivation of the wand system. The conversion of p-hy- droxyphenylacetaldoxime intop-hydroxyphenylacetonitrile is formulated asa simple dehydration reaction (Fig. 1). However, the reaction requires NADPH (2,4,26) and may be of complex nature.

The catalysis of the reaction sequence from tyrosine to p- hydroxymandelonitrile by two separate enzyme systems is also attractive from an evolutionary point of view b e c a w aldorimes are belioved to be the branch point from which either cyanogenic glucosides or glucoainolates are formed (27). No plant has yet been shown to contain both classes of compounds, but the enzyme ayetern catalyzing the bioeyn- the& of the lldoxime may be simikr in those p h t n which produce cynnogenic glucddes or glucmiwktee.

Detailed analyaia of the catalytic properties of the sorghum

00000001.tif00000002.tif00000003.tif00000004.tif00000005.tif00000006.tif00000007.tif00000008.tif