Studies on enzyme inhibition by fluoromalate and fluoroacetoacetates

ARCHIVES

OF

BIOCHEMISTRY

Studies

ANU

BIOPHYSICS

278-287 (1960)

90,

on Enzyme Inhibition by Fluoromalate and Fluoroacetoacetates’ EMERT

From the Department of Pharmacology, Research Lahoratory, Veterans

M. GAL

School of Medicine, Cniversit!l oj California, and Neurochemicul Administration Hospital Sepulveda, Sepulveda, California Received

May G, 1960

The inhibitory action of fluoromalate, fluoroacetoacetates, and fluorocitrate was studied in pigeon liver particulate, rat liver mitochondrial, and isolated enzyme systems. The results show that fluoromalate and fiuorocitrate increase acetoacetate formation from pyruvate and CO2 with concomitant depression of citrate synthesis in rat liver mitochondria but with an accumulation of cit,rate by the pigeon particles in presence of fluorocitrate. Fluoroacetoacetates arc inhibitory to both acetoacetate and citrat.e synthesis, particularly in rat liver mitochondria. Kidney cortex but not liver homogenatesrapidlyhydrolyze the fluoroacetoacetates tofluoroacet,ate.Considerable inhibition of the malic enzyme by fluoromalnte and 2,+fluoroacetoacetate was observed, while none of the inhibitors affected oxalacrtate decarboxylase. Only fluomalic dehydrogenase. romalate inhibited oxalncetate carboxylase zmd DI’S-dependent Fluoromalate, fluorocitrate, and fluoroacetoacetates inhibited fi-hydroxybutyrate oxidation, but only the fluoroacetoacetat.es inhibited aignificant.ly t.hc acetoscet.atesynthesizing enzyme. Acet.othiokinase, fumarase, ant1 isocitric (Tl’N) and srlccinic dehydrogennses were not inhibited by any of the fluoro compounds. Several aspects of the kinetics of inhibition have been examined. INTRODUCTION

In an earlier paper (1) it was demonstrated that, fluoromsla.te (FM)” hut. not. fluorocitrate (IX) inhibited the triphosphopyridine nucleotide (TPK)-dependent mnlic dehydrogenase in washed particles of pigeon 1 Supported by grant B-1159 of t,he U. S. Public Health National Instit.ute of Neurological 1)ise:tscs and Blindness, and by a grant from the American Cancer Society and by Cancer Research Funds of University of California. 2 The abbreviat,ions used are: FRI, fluoromalatc; FC, fluorocitratr; 2,-ImF.4cA(*, et,hyl %,l-fluoroacetoacetate; ?-FAcAc, ethyl I-fluoro:tcet,oacetate; 08, osalacetate; BOH, p~h~dros\l)r~t~raCe; AcAe, acetoacet,ate; TI’N (oxidized) and TPNH (reduced), triphosphopyridine nucleotidr; 1)PN (ONitiized) and DPSH (reduced). dir)hosphopyridirle nucleotide; ATl’. adenosine 5’.tril)hosphate; ITI’, inosine 5’-t,riphosphate; IDP. inosine 5’.diphosphat,e; CoA. coenzyme A; Tris, tris(hydrosymcthyl)aminomethane; GSH, redured glutathione; PEP, phospt~ocnolp~r~lv:Lte.

liver and partially purified mulics enzyme of chicken liver. This was contrary t,o the findings of ot,hcrs (‘2) who found 1% at, 3 X 10~” :I1 collrent,ratioll inhibitory to the ubovc systems. Revent’ repor& (3) indicate that much higher concentrations ( 10P3 to lo-” J1) of IX arc necessary to produce ahout, 50 7% inhibition of TI’K-dependent malic dehydrogennst. .b inhibition of diphosphopyridine nwleotide (DPX)-dependent rnalic dehydrogenase hy FM (1) as well as hy P-fluorooxaloa~eti~ acid (1;0=2.4) (4) was also noted. This paper is cwncerlled with a detailed presentation of the studies on the iuhihitory propertics of FM and fluoronreto‘u’c’tates. ‘ FSI’E’RIMFYTAL ,A 3 All chemicals were of reagent grade. Sodium fuoromalate was prepared by a method of Taylor and Kent, (5); triethyl flnorocitrate was synt’hesized according t.o R.ivett. (61, isolated as sodium salt (i), and prvified as report.ed (8). The ethyl

2%

cstP1.S of 2,1-fll~oroncet.o:rcet:lte (2,1-FhL4c)and -l-fllioro:lceto:tcetate (-2-F&&) were s~nt,hraizctl according to the methods of McBce et al. (9) and Fraser et wl. (lo), rrspectivel~. The TI’N and DT’SH, cornzymc 4, adrnosine triphosphabc (ATI’), and inosinc triphosphate (ITT) were purchased from Sigma Chemical Compmy (St,. I,ouis. No.), oxalacetatr (0.4) was obi,nincd I,!- the method of Hcidelberger and Hurlbert (11)) and the sotlilml p).ruvate from p?-rrlvic aci(l as given t,y 1totmt son (12).

The pigcon liver 1)urticles were prepared according to Iirebs and Davies (2), the liver mitochondria according to Hogeboom and Schneider (13). The kidney cort,ex (10% w/v) was homogenized in 0.9% KCl. Alalic enzyme and oxaloacctic cart~ox~lnse were purified from chicken liver (la), :~nd DPN-dependent malic dehydrogenasc, crystalline fumarase were purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio). l’artially purified isocitric dehydrogenase was kindly provided by Dr. Ii. H. Ibsen of the Universit,v of California at Los Angeles, and the condensing enzyme was a gift from Professor Sever0 Ochoa of the Yew York University. Succinic dehydrogenase was prepared from pigeon heart and breast muscle from pigeon liver (16). (15), arid acctothiokinase The :Icet,oacrt:tte~s~nthesizirig enzyme was prepared from pigeon liver (17), but was not carried beyond the ammonium sulfate fractionation step. Trsnsacetylasr was isolated (18) from Escherichia coli (strain 4157) kindly given by Dr. J. hliller of the UniversitJT of California.

The &oz (N) values were determined by Warburg direct method at, 3i”C., oxygen as the gas phase. Each Wnrburg flask, unless otherwise stated, cow tained 50 pmoles potassium phosphate bufl’er at pH 7.4, 4 pmoles iVgCln , 230 pmoles KCl, 4 /*moles ATP, or as indicated, and 1 ml. of tissue suspension. In the experiments on oxidative phosphor?-lation, t,he conditions were those described elsewhere (19). In the experiments 011 the effect of FM on oxaloacet,ate cnrboxglase, each flask contained 60 pmoles OA, 2 hmoles ITP, 2.5 pmoles NH, 2 pmoles Mn++, and 50 pmoles KaHC’403 (containing 2.7 X IO5 courits/min. of radioactivity) anti 15 units of enzyme, total v01111ne 1 ml., gas phase nitrogen, duration 5 min. The number of micromoles of fixed C140~ in the p~carbox~l of OA was determined by the known method (14). The barium carbonate samples were count,ed in a Nuclear Chicago D-47 flow counter. All samples were counted to 25,000 preset counts. Corrections wcrc

made for background and self-al)sorptiori. Assays for ‘IT&dependent malic deh~drogrnasc and oxaloucetic decarbos~lase were essentially those reported in t,hc literature (20, 21). The DPN-dependent malic tlehytlrogenuse Assam- leas that of Mehler ct ul. (23) with the enzyme activity- given for A log Ioil for the 30-&sec. reading at 310 mp, lvhile the effect on the condensing enzyme was measured I,?; determining acetJ-1 Ir~drosaniate (23). This met,hod was also applied in measluing changes in the acetothiokinase activity. -411 the optical measurements were carried out on :L Beckman DK-2 recording sprct,rophotometer. Sitrogen determinations were done by micro-Kjcldahl. Acetoacetate cletcrminations M-ere made by the method of Walker (24). Citrate was determined as described elsewhere (7). Inorganic phosphate was done by the method of Gomori (25). Protein was determined by the biuret test (26) or 1)~ the spectrophotomctric method (27).

Michaelis constants obtained in some expcrimerits were determined graphically t,y the method of Lineweavcr and Burk (~8)~ while the k’r values were obtained by the method of Dixon (29). The hydrolgsis constant,s, KB, of the fluoroucetoacetates were det,ermined by measuring the drcreasc of opt,ical density at their X,,,x against time. The kinetic equation for the reaction of first order yielded t,he hydrolysis constants (Table I). RESULTS

In

at,ternpting

to evaluate

the

inhibitory

effect of fluoro cornpouuds, the degree of purity of these compounds is of great irnportance. This is particularly significant in t,he case of synthetic ET, since the chemically pure triester of fluorocitric acid cowtaim several chromatographically discerni-

280

EMERY

M.

GAL

in either syst)em by FC and not wry significant with FM. However, 6.6 X 1OP ~11l+‘C and 0.6 X 1OP 31 PM will produce 50 % inSystem as under Esperin~ntal. .411 flasks conhibition of the respiration. The total amount) tained 2.7 X 10F2 M pyrnvate and 1.2 X lo-* L%l of pyruvate expended in both syst,ems KHCO, The inhibitor roncentrations were 6.6 X through respiration, AcAc, and citrate forlo-” !I[ except FC, which was 3.3 X lo-” M. mation is very nearly equal. The controls gave about 13 pmoles in the pigeon liver part#icles and about, 11 pmoles in the rat, liver mitochondria. The addition of 1.313 X lo-’ ilr fumarate to t)he system, in presence of ICC, given in Table II, increased &rate awumulation to 9 pmoles/mg. X in the pigeon liver mLT,’ nGiesi 7nL,f’ dLT/ 1NY. LV’ ,,ig.3’ 1mg..v wig.4’ particles and to 12 pmoles/mg. N in t)he liver mit)ochondriu, with the contSrol figures being None 179 3.11, 1.65 188 0.91 2.60 FC 1101 3.91 3.26 125 2.91 1.6-l 3.1 and 9.3 ~moles/mg. K, respectively. This FM ’ 176~ 3.70 0.56 148 2.03 1.63 is in dircc+ contradiction with the observa2,4-FAcBc 158’ 2.92 1.37 173 0.151 1.10 tions of others (2) who found that the adI-FAcSc 176’ 3.04 1.75~ 165 0.56 1.24 dition of fumarat’e did not, lead to cit)rate accumulation in the presence of FC. The inble components, most of t,hem, unlike I’C, hibitory effect’ of PM was reversed by about unable to produce citrate accumulation by 5 X lo-” -11 fumarate. It’ is interesting to not,c fhat the fluoroinhibiting aconitase (30). In many inacetoacctat,es are of no influence on the citstances (2, 3) the preparation of the sodium rate formation or awumulation in t,he pigeon salt of I+K” is different from that employed here. The most common impurities of syn- liver particle system, while t,hey are strongly inhibitory of cit,rate format#ion as well as acethetic fluorocitric acid samples are fluorotoacetate synthesis in rat, liver mitochondria oxalaret,atc and oxalic acid. In psrtiwlnte systems, fluorooxalacetate (6.6 X IOF to c~en though the inhibit,iou of respiration is uot) noticeable. In some of the experiments, 1O-3 M) produced about) SO%, and oxalatc when 12(aA(aformation against time was (5 X lop5 11) 20 % inhibit,ion of citrate synthesis from pyruvate and CO, , rcspec- measured in the presence of the inhibitors it was found to be linear (Fig. 1). The decreased Gvely. A4c;1(dformation from pyruvate alld COZ in The results of experiments on the inhibithe presence of fluoroacetoacetat’es indicates tory effect of some fluoro wmpounds with an interference at, the site of the .4cA4c-syw pigeon liver particles and rat liver mitochondria are presented in Table II. In both t)hcsizing mechanism. Confirmation of this is systems the effect, of 1X and FM was to demonstrated by experiments with puriincrease acetoacetate format,ion, which was fied enzymes (Table VII). The lack of &rate awumulatioll in presin keeping with the kctogenic effect observed in IGO (31). The fllIoroacet,oa~ctates are ence of the fluoroacctoa~etates indicated the markedly inhibitory of ucet,oacetatc (AcL1c) inabilitv of the 1ivc.r to utilize t)hem for b‘C synthesis in rat li\*er mitochondria, and at’ synthriis. This latter effect resembled the 3.3 X lo-” II1 concentSration the inhibition is observations of others (3”) on rabbit, kidney Howe\-er, ill homogecomplrtje. The difference in the abilit,y of the wrtex mitwhoudria. nates of rat kidney cortex they irot’ only t,n-o systems to synt,hevize citrat#e from pyinhibited respiration but proruvnt,e and CO? is quite striking inasmuch as powerfully the pigeon liver particales respond to FC by a duwd aocurnulation of citrate (Table III), twofold accumulat,ion of citrate, while ilr the indicating their rapid hydrolysis to fluororat liver mitochondrin F’c like FM pro- acetate (I’&) in the kidiley but Ilot in the liver. duced 40 % inhibition of citrate synthesis. The effect of E‘RI on oxidativc phosphoThe depression of respiration is only 35-40 % TABLE

EFFEC'T

II

OF FLUORO-CO~POI:NDS ENZYX~ 0~' P~RT~~:~I.ATE

ox THE SYSTEMS

RL4Lrc

ESZFME

IO

20

IXHIBITION

30

40 Time

Frc:. 1. Formation

of AcAc from pyruvatc

50

60

70

(mln)

nntl CO?

Conditions

as given in Table

II.

rylation was examined and found to be similar to that of FAc (19). During the course of these st,udits on oxidntive phosphorylat,ion it was not,ed that the inhibition of citrake synthesis produrrd by E’C but not by FM in rat liver mitorhondria was considerably reduced by t,hc addition of glucose and hcxokinasr to the system described in Table II. In fwt the inhibition could be cwmpletely overcome, by adding ICI’, increased amounts of .\Tl’ and phosphate t,rapping system, to the, point of ensuing citrate acwnnulutioii (TLLblP TV).

III an earlier communication (1) widcnce uw presented that, FC e1’en at 6.6 X 1OF -11 c,ollc,rlltr.:ltit,II did notj illhibit’ malic ellzymc ; consequent Ip \v(’ have assumctd that the (i!) % inhibition observed by others (2) at, j X IO-” A/ c~on~ciitrnt,ioll was by a substanw other than ICC’.I’rom the experiments prcantt>d ill Tattle V, it is appnwnt that)

l~luorwitrate at 6.6 X lo-” M cvnctntrat ion was only 22 5%inhibitory, I\-hilt at the same c,ollc.entrat,ioll lJh1 produwd 60 % or more inhibition. In i he experiments where the elkzymc was preinrubuted with I’C‘ :il~d 1’1’s before addition of 50 Fmoles malate, 6.6 X 10e5 :I/ c.oncwitr.:itiolI of FC’ wa.q sufic+nl~ to (YIUW 19 5%illhibitioll.

TABLIZ 1iEVERSAL TION

IV

OF F~,LI~R~~~~~~R.~T~-PRoI)u('ED OF CITRSTI~ STSTHESIS 1s IdIVER ;\~ITOClIONl,RI.4

IYIIIBIIt.4~

System as in Table II except that all Hnsks cwtained additionally: 60 pmoles gllwose, 0.56 mg. (92 K.M. units) hesokimtsr, ATP, :~ntl KF unless otherwise indicated. FC concentration ww 3.3 X 10y5 M. The results are averages of at least triplicate determinations. Ratio of citrate FC; control

Earlier it was also noted (1) that I;,14 n-as inhibitor of TPK-dependent malic dehydrogenase, and kinetjic st,udies confirmed t’his. The competitive nature of I;?II against’ L-mslate is graphically represented in Fig. 2. The inhibition of malate oxidation by I;31 at eight levels of L-malate (lo+ to 2 X 1OP 31) at varying concentrntions of the inhibit,or yielded a k’, of l.O’i X lo-“. The graphical method (29) of determination of the K, of L-maMe (plot of S: 1’ against, S) under the experimental conditions ga\~ 5 X lo-” AI, which seems to hc in good agreemcutj with the value for t hc pigeon liver enzyme (20). Similarly, the effect of 2,-LFdcAc on this enzyme It-as also rxnmined as sho\vn in Fig. :2. The value of KI for 2 ,l-FAcXc was 1.7 X 1OY. At similar inhibitory c.ollcentrations, the l-FAcAc had no effect on this enzyme. It was found in agreemcllt wit,h others (3, 33) t)hat the prevailing concentrat~ions of Mn++ determine t)he extent, of inhibition. a competitive

EFFECT TABLE IXHIBITION

OF

bIhr,rc

\’ l:IxzYhiE

BTi

FLUOROCITRATE ANI) FI~COROMALATE The system contained: Tris buffer pH 7.4, 0.02 M (1.32 X 1OW %); XInClz (2.0 X 10-l 111); Tl’r (1 X lo-’ 211); malic enzyme (C, gel elated) 0.1 ml. (equivalent to 6.4 pg. Iijeldahl nitrogen); total volume 3 ml.; t,emperature 30°C. The malate concentration in the cell watts 5 X lWz 31.

dl

OK OXALACETATE

DECARBOXYLASE

An esaminat,ion of t’he inhibition of the Rln++-activated oxulacetnte decarboxylase activity by fluoro inhibitors revealed that FC produced 20 % inhibit8ion only at 6.6 X 10-h Al, whereas 6.6 X 1OP II1 FM was required for the same degree of inhibition. The fluoroacetoacet,ates had no eflect, even at much higher concentrat,ion. In all the experiments, the spont,aneous decarboxylation of Oh was also measured simultaneously without the presence of the enzyme, in order to correct for the actual enzyme activity. The prestncc of the inhibitors did not seem to have any effect, on the nonenzymic decarboxylation of OA.

(a)

x 10-d X 1OW x 10-a FRI x 10-s (b) Preincubat)ion of enzyme : with 0 Control 5 0 FC 3.3 x 10-j 5 3.3 x 10-j 0 6.6 x 10-s 6.6 x 10-j 5 Control FC

3.3 6.6 6.6 3.3

25.0 21.5 19.5 10.0 inhibitor: 18.6 16.4 20.7 14.6 20.9 10.6

14 22 60 100 12 29 49

EFFECT 0~ ~~rrorio ~SHIBITOM~ ~)xALACETATE C.lltBOXYL.~RE

ok-

In the light of the inhibition of enzymes catalyzing react’ions involving malatc and OA by fluoro compom~ds, it was of interest to examine their influence on oxalacetate carboxylase. The i&opic incorporation of CO, into OA according to the reaction scheme : OA + ITP

Ml1 + @

PEP

+

ID2

+

COY

ENZYME

‘28::

IiYHIBITIOX

2

4

6

8

IO 12 14 16 18

(1) XlO-5 FIG.

2. Inhibition

of TI’K-dependent

I

: ’ KI

3. Inhibition in Table V. FIG.

malic dehydrogennse

g FM

by FIT.

Coutlitions

5x d

v L-molaie

1

I I

I 7 x lo-4

of TPr‘;-dependent

(I)

malic

dehydrogenase

. x lo-4

&j

by 2,4-FAc$c.

as given

in Table

V.

I 2 2,4-FAcAc

Conditions

as

given

28-l

EMERY

was considered a more reliable measure of actirit,y t,han the reverse reaction measuring OA formation, because in the latter tho determinations arc vitiated hv the interference of the fluoro inhibit,ors. Strickland (3), who used this lat#ter approach, found FM fully inhibitory at, I OF JI conwntratjion but n-ithout any cffwt at IOP 11 wit’h lo-” dl phosphoenolpyruvnte as wbstratc. He also found 20 7%inhibition with ICC at 1OF III c’onc’entrstion. The experiments here reportjed and summarized in Table \‘I show that above lo-’ J/ concwtration the inhibition rapidly TABLE b:FFE(‘T

OF

Inhibitor

UN

C.4RBOXYL.4SE

as under Experinzenlal.

concentration x

Inhibition

l+Yxed CO?

103 .sr

None 0.6 3.3 6.6 12.0

GAL

irwrearea in a nonlinear fashion. For the linear portion of inhibitor concaentration [I] versus 1/Ti the Kr of E’RI was found to be (i X lo-“. However, the inhibition was about, 80 “; , e\yen at 1.2 X IO-” JI ~onwnlratioi~. At 6.0 X IOF ~11cwwentration of FC, 110 effect was found. Similarly, the fluoroacct owetates displayed no inhibition.

I’reviously (1) it’ was mcntiolled inhibited the reaction: OA + I)PSHy

h(‘OROMALATE

()XAL.ICETATE

Conditions

VI

M.

plllole.~

“:L

0.75 O.Tl 0.64 0.46 0.15

Sone 5 15 35 80

. : m:tlate

that 1cM

+ 1)1’S

The rate of 0.4 rcduc+ion was examined beWeen lop5 to lo--” X concentration at pH 7.1 and WC., where the Ks value of OA obt,ained was 9.8 X 10-j. This is somewhat different from the 2.6 X 10M5at pH (i.7 ob served by Davies and Kun (34). In E’ig. 1 the graphical rcprrsentatjion of the competition between OA and FM is given. The K, found for the FM v-as I X 1OF. The enzyme inhibition is 80 ‘34at 6.6 X 10~” :I/ of l:XI. Con-

40 -

1 V 30 -

FIG.

pmole;

4. Inhibition of OA reduction by I>PKH. Conditions: enzyme, Tris buffer (0.02 N), pH 7.4; temperature 27°C.; volume, 3 ml.

4 pg. prot.ein;

DPKH,

0.3

firming 1hc observations of others (34), the kink studies wit#h OAAabove lo-,* JI (wnwlltrat,ion xwe complicated by the itlhihitory action of OA itself. It n-as found that the coupled reac+on of DPK-malate hy malice dchydrogenase with condensing cnzyme altd acetyl-CoA (35) (or lvith acetyl phosphate and transncetyluse) will yield a mow reliable Kl value for IW, csptcislly as c~sprrimrnts showed no inhihitSion of either thr cwndensing enzyme or the transacetylase 1)~ km :at lo-” ;1/, :I c~oncwitratioii cornplrtc~ly inhibitory to mnlic dehydrogenasr. (‘ant rary to the earlier report, (l), further expcrimclrts wit.h 2,4-F,k.Q as xc11 as with 4-1:.-\(*.1(~showed no inhibition of the cuzymt~.

It 1~x8 mentioned carlicr t,h:lt I:(: and liY1 iti p:n%cwlatc~ systems incwasetl Ac*hr format ion from pyruvatc and (‘0.’ I\-hile fluori11n1cdawtoacetates depressed it. rt wasof iiltcwst thcrcfort to cxamiiir their effect on part ially purifkd nc~to:lcetat,ct-syllthrsizillg cilxyme from pigeon liver. I’igeoil li\yer was c~llosc~11 twauw it showed itsrlf to bc less nffwt cd by fluoro:lc,ct,o:tc~ttatcs (Tahlc II) t ban rat liver mitochondrin. I’rom Tnt~le \‘II it is apparcvrt that I’l\I did not inhibit Ac.1~ synt lirsis cveii at 1.3 ni,l/ c~onc~eiitratioii, n-hilt the fluoro:L~eto:l~et,~~t~sat this wl~wlltration produced c*t~iisider:it~k iiihihit ioii. .I con-

plctc inliil~it~ioll owurrcd at about IO m.lI ~oiic~c,iltr:itiolls of the fl~loro:ic~Ctoac,~~tat~~s. Thtsc ohservat ioirs are cwnsistcnt t hrrcforc \vil h t how found in t,he mow c~m~ple?;partic&lllntc or mitoc*ho~ldrial prqxtratiol)s. Iteversihility of the inhihitioll prodwed by fluoroacfietoawtnt es \vas not c~splorrd. tlon-r~~~~r, there is indirwt evidence from the studies of thrir effect OII P-ll?ldroxyl)utyr~~tc (BOH) osid:~tion ill rat liver nlitoc~hot~cl~~i:~ that illcrcawd 13011 c~o~ic~eiltr:It.ioII(311diminish 1hc . degrw of ~l~hll)~tNM brought- al)0111 l)y fiuoroawtoawt atw. Thv rwl11t s of the inhihitiolr of ISOH osidatioll arc prwe~ltc~tl ill Table nrr. It is of interM to nok that FM aIs0 iilhil)itcd I%()I1 osiclatioll already at 0.6 mJI c*ol~cwltratio~l IU&Y the gi\wl qwimclltal c*olltlit ioiis. h sonic pwlimi~rary csperimttiits, l~~wc~~~r, it \\-a+ ol~wr~wl that atlditioil of glucosc~and hesokin:ts;c ~1strapping s\rstcm :uld 1 +~olt~ .17’1’ to t lw syst~~n g11~(wiii T:i~~l~~\‘I II c~nnwlctl ~lic iilhil)itorJ dfect of I>LI 1111t riot that of the fluorc,:l~cto-

acetates on I30H oxidation. This is now under further investigation. In a series of experiments, the effect of FM and fluoroacetoacetates on several enzymes n-as examined. The following enzymes showed no sign of being inhibited: aretothiokinasc, fumarase, a&l isocitric: (TPS) and swcinicb drhydrogennses.

The elegant work of Morrison and l’eters (38) is a good example of real&G evaluation of awnilase inhibition bv 1X at, 2.1 X 10-j M l:C cqonccntration, with the ratio of (*it,rate to FC being 100: 1. In the case of FM the effective inhibitory c‘onccntrations used in ~ilro arc much smaller t,han those tried i?l l’iL’0 (31). E’1\4like FC is ketogellic:. This effec% is easily understood from the inhibit)ory property it exerts on both t,he enzymes involved with pyruvate oxidat)ion t,hrough the rewtiow :

The apparent, impairment of citrat’e synt,hesis from pyruvate and (W2 by the rat, liver mitjochondria in the presenw of l’(’ at 1’P:P + Il>l’ + CO?.d Oh + ITI’ ((1) t3.3 X IO-” X concent,ration show a no& wort,hy contrsst~ to that, of t,he pigeon liver pyn~vate + COl + Tl’PI:H2; Amalate + ‘l’PK (b) particles prepared according t,o Krebs and Davies (2). At, t,his concent,ration PC did not as well as through the inhibition of produce any inhibition of the purified malic AcAc + DE’KH2. ’ BOH + III-5 Cc) enzyme, suggesting that t,he inhibition of citrat,e synt,hesis by 1X in t,he presence of but not pyruvat,e and CO1 may rest with it,s effect 2 Acet~l-CoA ti AcAc + 2 Co,4 Cdl, on phosphonuclcotides. Indeed, as we have demonstrated elsewhere (X), the dephos- On t,he other hand , 2,4-FAcAc, while it is inhibitory to reactions (b) and (c) is also inphorylation of adenine and pyridine nucleotides by rat liver mitjochondrix in t,he pres- hibitory to reaction (d), hence there is a decrease in Acdc as \I-ell as in citrat,e syntheence of P’C was significantly increased. sis. The 1-I:Xc.4c behaves very much like the Furthermore, t,hc experiments (Table IV) demonst,rated t,hat. addition of I<]+‘, increas- 2 ,I-FhcAc except, that, it, does not produce any block of citrate synt#hesis in the pigeon ing amount of XTI’, and a phosphate-trapping syst,em reverses inhibition of citrat)e liver part,icle system. The lJh!I and fluorinated acetoacetates are reversible inhibitors, synthesis from pyruvate and CO2 in rat, liver of the enzymes studied. mit,ochondria. This is (so&rued as an indiIt is perhaps profitable at, this point t,o cat,ion t,hat FC is not directly inhibit,ory to speculate t,hut. the significant inhibition of the mslic enzyme. both AcAc and citrat’e formation by the fluThe results of experiment’s with purified enzymes demon&ate that, 1+X below 1OV A/ orinated acrtoacetates in the rat liver mitoconcentration is not, an inhibitor of these en- chondria might, allow somr explanation of zymes. The observatJions of Stickland (3) t,he failure of t)he rat liver in ~ivo t,o activate clearly indicate that low3 to IO-’ 31 concen- IF;&:. The cnzymic conversion of fluorotrations of FC are required to produce GO- awt.yl-Co.4 t.o 2 ,A-I:Achc, which was indi80 5%inhibition of the malic enzyme with the cated by 13racly (59), may suggest, that a ratio of malat,e to 1°C being 1: 1 or 1: 10. In series of possible reactions call take plac*e order to ascribe any significance t,o the in- between ncetyl-CoA and fluoroacetyl-Co.4 hibitory action of a compound vis-A-vis an leading t,o possible acetoacet,at,es such as the 2- and the 2 ,L or 4FXcAc. lcolloming their enzyme, it is necessary that’ t,he compound should inhibit the enzyme concerned in synt.hesis in the liver they could account, for vitro at concentZrations consistent wit,h those a lack of ketone body formation which one effectively inhibitory or toxic in U~PO.It, is notices aft,er FAc administjration. Obviously this could not, happen if 1~Ac were to go to therefore questjionablc whet’her such inhibitjon by FC is of any metabolic significance at the ket,ogenic FC. Furt,hermore, recent oball, since 0.05P0.10 pmole of synthetic ICC servations from in LGO studies wit,h C14-lacan effect,ively block citrate oxidat,ion of t’he beled 1cAc gal-e strong evidence of incorpobrain kjoth in ciw (31) and in ZY&W(:37). ration of label into t.he different fatty acid

EKZYME

fractions of t,he rat liver (40), thus also indiwting a pzLthwsy via XcAc synthesis. I;inally, the fact that the fluoroacetoacet,ates w&k hydrolyzed tjo FAc and in turn :tctivat,ed to E‘C by the homogenate of rat’ kidney corks (Table III) hut not t’hat of t’he liver support,s the conwpt of FAr detoxicxtion mechanism through fluorinated acetoawtutes as was originally proposed hy Haand Woodmard (II). ~CYI, Ilamsey, ‘l% author is grat,cful t,o Mrs. Karen hInt tingly, Mrs. Edith Roth, Miss Patricia Drewes, and Mr. Attlin Huish for their skillful technicnl assiatmce. REFERENCES Hiophys. 73, 279 1. (;,\I,, 12. AI., .trch. Biochem. (1958). 2. KRERS, H. A., AND ~>.~vIEs, I). I)., Arch. xi. biol. (Bologna) 39, 533 (1955). 3. STICKLASD, R. G., Biochem. .I. 73, 646 (1!159). -1. KGS, E.,(:RASSETTI, I~.K.,FANSHIER,I).\li., AND FEATIIERSTOXE, R. ?*I., Biochem. r7Lacoz. 1, 207 (1959). 5. TAYLOR, S. F., ANT) KENT, 1’. W., J.

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