THE STICKLAND REACTION · obligate anaerobes, the supposed toxicity of acceptor amino acid in the...

THE STICKLAND REACTION

B. NISMAN

Institut Pasteur, Annexe de Garches, Garches (Seine et Oise), France

Introduction.................................................................................... 16Stickland reaction studied with cell suspensions.................................................... 16L-Amino acid dehydrogenase system............................................................. 21a-Keto acid dehydrogenase system............................................................... 29Acceptor amino acid reducing system............................................................ 37

The enzyme systems responsible for the oxi- be hydrogen donors. Among these were: alanine,dative deamination of amino acids have been valine, leucine, isoleucine. Other amino acidsextensively studied in higher organisms, in some were found to reoxidize reduced phenosafranin,facultative anaerobic bacteria such as Proteu benzylviologen and methylviologen in the pres-vuljaris and Proteus moranii, and in some fungi ence of a suspension of the same organism; thatsuch as Neurospora crassa [cf. general reviews by is, they were shown to be hydrogen acceptors.Krebs (1, 2)]. These enzyme systems are char- Examples of such amino acids were: glycine,acterized by the use of molecular oxygen as a proline, hydroxyproline, ornithine. Sticklandfinal functional hydrogen acceptor. With the then showed that the interaction of a donor andobligate anaerobes, the supposed toxicity of acceptor amino acid in the presence of a bac-oxygen for these organisms has somewhat re- terial suspension results in a mutual dominationtarded the studies on the oxido-reduction en- as determined by ammonia formation.zymes. Only recently has work on the enzyme As examples of such coupled deaminations wesystems, involved in the coupled deanination may cite alanine + proline and alanine +discovered by Stickland (3, 4, 5, 6) and Woods glycine; the over-all reactions can be represented(7), been taken up again. as follows:

CHCHNH2C00H + 2 NH2CH2COOH + 2H20 3 C113COOH + 3 NH, + C0,alanine glycine

HC CH2CHCHNHCOOH+2 l l +21H20 - CHsCOOH+2NNH1(CH,)4COOH+

alanine H2C C11C00HNH3 + CO,

NHproline

THE STICK.LAND REACTION (SR) STUDIED Starting with a donor amino acid and follow-WITH CELL SUSPENSIONS ing the reaction stepwise one may represent it

Coupled deamination between two amino in three hypothetical stages as shown at top ofacids acting as donor and acceptor of hydrogen page 17.respectively (3, 4, 5, 6) is the principal chemical Woods (7) was able to show that both the Dreaction by means of which C(o8tridium 8poro_ and L forms of proline act as acceptors. Usinggenes and other bacteria (8) obtain their energy C. sporogenes suspensions, Kocholaty andwhen growing with amino acids as sole sources Hoogerheide (9, 10) made detailed studies ofof carbon and nitrogen. Stickland first observed donor and acceptor substrates and also demon-that in the presence of a suspension of C. sporo- strated the reduction of proline and glycine bygenes certain naturally occurring L-amino acids molecular hydrogen in presence of a suspensionreduced methylene blue (MB), brilliant cresyl- of the same organism (10).blue and benzylviologen, thus showing them to Occurrence of the SR among the strict anaerobes.

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19541 THE STICKLAND REACTION 17

(a) CHsCHNH2COOH + H20 CHaCOCOOH + NH, + H,.

(b) CHsCOCOOH + H20 -k C0,COOH + CO + Hi.

(c) 2NHCH2COOH +2H -+ 2 CE4C OOH + 2 N11s.

HXO-CH2or (c') 2 l l + 2H - 2 NE6CHC1CH2CH2C0OOH.

HC CHCOOH 6aminovaleric acid

NHproline

Almost all the bacterial species acting on amino and also C. propionicum (16) which fermentsacids by coupled deamination have been shown alanine, among other amino acids, without theto belong to the family Clostridiae, as defined by presence of any added hydrogen acceptor:Pr6vot and others (11, 12, 13, 14, 15). 3 CHCHNH2COOH + 2 H20A. Proteolytic clostridia which effect the SR

3 CHsCH2COOH + CHsCOOH + 3NH,Clo8tridium botulinum C. sordelii There is evidence that certain rumen bacteriaA and B C. sporogenes can oxidize amino acids to the volatile branched

C. bifermentans C. valerianicum fatty acids possibly by way of the SR (17, 18),C. butyricum C. aerofoetidum but the responsible bacteria have not beenC. acetobutylicum C. carnofoetidum identified.C. caprowum C. mitelmanii Products of the SR. Stickland (3, 4, 5, 6)C. histolyticum C. ghonii identified the following end products of theC. saprotozicum C. indolicus coupled demination of alanine and glycine or

B. Nonproteolytic clostridia which do not effect proline:the SR Alanine acetic acid + NHs + C02.

Clostridium iodophilum Welchia perrinens Glycine acetic acid + NH,.(C. welchii) Proline 6-aminovaleric acid.

C. saccharobutyricum Plectridium tetani Using valine, leucine or isoleucine as hydrogen(C. tetan- ) donors and proline as acceptor, Cohen-Bazire,C. tetanomorphum In~fla( teras) Cohen and Pr~vot (13) obtained the following(C. term) branched volatile fatty acids with suspensions of

One may add to the previous list of bacteria C. caproicm or C. valerianicum:not effecting the SR, Diplococcus glycinophilus Valine -- isobutyric aciddiscovered and shown by Cardon and Barker Leucine -- isovaleric acid(16) to ferment glycine nonreductively, Isoleucine --+ valeric acid, probably

4 CH2NH2COOH + 2 H20 the optically active form.4 NH, + 3CH0 COOH + 2 C02, The reaction equations may be written as follows:

(CH3)2CHCHNH2COOH + 2 + (CH,)2CHCOOH + C02 + NH, +vCOOH

NH NH2(CHE)4COOH

(CH,)2CHCHCHNH2COOH +22+21- (CH,),0HCHCOOH + NH, +% COOHNH C02 + NH2(C0H2)4COO

CHCHCH2CHNHCOOH + 2 H +2f 0,C1100CH1CHCOOH + NH, +jCOOHNH CO + NH2(CH2)4COOH


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18 B. NISMAN [VOL. 18

TABLE 1 deamination occurs by means of hydrolyticOxidation of amino acids by Clostridium deaminase, and it is the resulting keto acid

sporogenes suspensions which is dehydrogenated, a reaction we do notNO CATA-UZ nUS C&TAXASIC

intend to discuss in this review. Catalase doesNO CATALASE LUS CATALASEnot influence 02/NH, ratios for any of theseSUBSTRAT- + a- + a- amino acids (21, 22) (table 1).

-Os +NH& keto -O2 +NHa ketoacid acid Products of the aerobic oxidation of the aminoacids. Nisman and Vinet (21, 23) have shown thatAlanine 44.6 49 1.2 46.1 51 1.6 the aerobic oxidation of amino acids of group 1

Valine 44.4 52.2 3.8 46.8 51.7 218 results in the formation chiefly of volatile fattyIsoleucine 21.6 24.2 3.2 23.8 25 39 acids together with small amounts of a-ketoDL-Norleucine 42 47 6 43 48 6.2 acids. The following a-keto acids have beenDL-Methionine 19 40 - - - found (as characterized by formation of 24Phenylalanine 5.4 22.7 3.7 - - dinitrophenylhydrazones (21, 22, 23):DL-Threonine 24.4 39.9 6 - - -DL-Serine 20.2 49.3 - - - _ Valine -* a-keto a-methyl butyric acid

I______I__________- - Leucine -. a-keto 'y-methylvaleric acid50 I'M of L-substrate in phosphate buffer, m/15, Norleucine -- a-ketocaproic acid

pH - 7.1 + 1 ml bacterial suspension (6.1 mg N); Isoleucine -- a-keto fl-methylvaleric acidin center well 0.2 ml 20% KOH; final volume3.2 ml. Gas phase, air; temperature, 37 C; time, The principal end products of the oxidation are120 min. All results in pm and corrected for volatile fatty acids.blank. Alarune a acetic acid

Valine isobutyric acidThese volatile fatty acids are the normal end Leucine -) isovaleric acidproducts of the metabolism of proteolytic Isoleucine -a valeric acid, probablyclostridia growing on amino acids or protein sub- the optically active form.strates (13). But if the same organisms aregrown in the presence of an excess of glucose, As can be seen, these are in fact the same fattyno branched volatile fatty acids are formed in acids as are formed when the hydrogen acceptorthe cultures, the products being then butyric is a "physiological" one (acceptor amino acid).and acetic acids, resulting from carbohydrate It has also been shown that under aerobic con-fermentation (19). The above mentioned ditions, clostridial suspensions are able tobranched fatty acids have been shown to be decarboxylate oxidatively the a-keto acids of theformed from amino acid fermentation in the donor amino acids (21, 24).rumen (17, 18). Analysis of the fatty acids pro- The following reactions occur in the presenceduced has been carried out by the very specific of the amino acids and oxygen as electronmethod of gas-liquid partition chromatography acceptor (the same is also observed in anaerobesdeveloped by James and Martin (20). not carrying out the coupled deamination):

Classification of the amino acid donors. The R-CHNHCOOH + X 0,amino acid donors may be divided into two -- R-COCOOH + H20 + NH3.groups: 1. The aliphatic amino acids which areat an oxidation state two hydrogen atoms more RCOCOOH +X 02 RCOOH + 00,.reduced than the keto acids: alanine, valine, Competition between oxygen and amino acids asleucine, norleucine and isoleucine. 2. The amino hydrogen acceptors. Experiments were carried outacids which are at the same oxidative state as (23, 24) with a system consisting of an aminothe keto acids: serine, threonine, cysteine, acid as hydrogen donor and oxygen as a hydrogenphenylalanine and methionine. acceptor, to which was added a second acceptorThe ratio of oxygen consumed to ammonia such as glycine or proline. In the presence of two

liberated is 1:1 for the amino acids of group 1; acceptors one might expect a decrease of oxygenfor those of group 2 the ratio 02/NHs is about uptake equivalent to the amount of the reduced0.5/1 with the exception of phenylalanine for amino acid acceptor. Such indeed was observedwhich it is about 0.2/1. In the second group the in the following systems: (a) alanine + glycine +


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1954] THE STICKLAND REACTION 19

TABLE 2 the reduction depends on the relative efficiencyCompetition between oxygen and glycine or proline of the amino acids used as hydrogen acceptors.

in the oxidation of donor amino acids by Suppres8ion of the phy8iolial acceptorClostridium sporogenes Suspensions actiity with arsenite. Arsenite has been shown to

mtJBSm1m +S- +I~l eTODB1TIONinhibit the SR (24) but not the oxidative de-xo&AON ID0,UPT amination of amino acids when the acceptor isBYM MM BY MM% oxygen. The system involved in the reduction of

50 alanine 47 54 0.7 the amino acid acceptor has been found to bevery sensitive to this inhibitor (24). The reduction

50 alanine + 29 85 1 39 of the acceptor can be shown to be suppressed100 glycine almost completely by sodium arsenite in a

reoxidation test using leuco-phenosafranin, or by50 alanine + 23 54.8 51 measuring molecular hydrogen uptake in the100 proline presence of glycine or proline (23, 24). In a

50 leucine 48 52 1.3system where both oxygen and amino acid arepresent, arsenite has been found to inhibit only

50 leucine + 27.2 95.1 2.2 44 the amino acid reduction, the oxygen uptake is100 glycine the same as in the donor system without in-

hibitor (see table 3 and figure 3).50 valine 45.4 54.8 Oxygen as a nonphysiological hydrogen acceptor.

It has been assumed that strict anaerobes possess50 valine + 14 52.8 - 67 no enzyme systems capable of using oxygen as a100 proline hydrogen acceptor. Nisman et al. (21, 22, 23,Each Warburg flask contains the substrate in 23a, 24) and Aubel et al. (25, 26) have shown

phosphate buffer (0.004 m, pH 7.3); in the side respiration among anaerobes, i.e., oxygen as aarm 1 ml bacterial suspension (4.76 mg N). The hydrogen acceptor, in the oxidation of aminoenzyme is added to the substrate at 0 time. All acids and other substrates by suspensions ofthe other conditions are identical to those ex- many clostridia. Interestingly, StadtMan andpressed in table 1. Barker (27) independently and almost simul-

taneously demonstrated that oxygen is used by aoxygen; (b) valine + glycine + oxygen; (c) strict anaerobe, C. cluyveri, in the oxidation ofleucine + glycine + oxygen; (d) alanine + ethanol. Enzymes using oxygen as an acceptorproline + oxygen; (e) valine + proline + have already been known to take part in theoxygen. anaerobic coupled deamination. If the two typesTable 2 and the figures 1 and 2 show that the of acceptor are present together in one system,

uptake of oxygen is 40-45% less for systems competitive reduction sets in.containing glycine as a second acceptor and 65% The enzyme system responsible for theless for systems containing proline than for the reduction of glycine and proline in the SR is acomparable systems in which oxygen is the only hydrogenase (28) and was shown to catalyze theacceptor. The amount of amino acid acceptor following reactions (29, 30):reduced corresponds exactly to the diminution of i. H, 2H + 2eoxygen uptake, one molecule of oxygen beingequivalent to two molecules of the acceptor ii. 2H + X XIIamino acid. The difference in the diminution of i + ii. 2H, + 0. = 2H20oxygen uptake between the systems containingglycine and proline seems to be due to the more The hydrogen acceptor for reaction ii may berapid reduction of the latter acceptor. To MB, thiosuiphate, nitrate, sulfate, carbonate,summarize, one may say that under aerobic C02, CO, fumarate. Only obligate anaerobes canconditions the amino acid acceptors compete use certain amino acids as hydrogen acceptors.with oxygen for the hydrogen evolved in the Farkas and Fischer (31) have studied thedehydrogenation of the donors; the total oxygen reduction of fumarate by molecular hydrogenuptake is accordingly reduced, and the extent of using Proteus vulgaris suspensions and have


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m/: 02 SQMt0QQ _-

1000/

800 4,

6'I

600 -2yo. Qi

200

0 20 40 608Ot/00 120 /60TIME IN MINUTES

Figure 1. Competition for the hydrogen evolved from leucine between glycine and oxygen acceptors.The conditions are those described in table 2. Curve A shows the oxygen uptake by the presence ofleucine alone. Curve B shows the oxygen uptake by the system leucine + glycine. The oxygen uptakeis corrected for the blank.

Ml. 002 _

1A000Sooo

600 2f 0 A14>

400 /

/vAiNS0.L'I.

200 /

2'o|| 2 I0 20 40 60 d010A AX A40TIME IN MINUTES

Figure 2. Competition for hydrogen of donors between oxygen and glycine or proline as acceptors.The conditions are the same as in table 2. Curve A: oxygen uptake in presence of alanine. Curve B:oxygen uptake in presence of valine. Curve A1: oxygen uptake in presence of alanine + glycine. CurveA2: oxygen uptake in presence of alanine + proline. Curve B1: oxygen uptake in presence of valine +proline.

20


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TABLE 3 against the peroxidative action of the ultrasonicSuppression of the competition between oxygen and vibrations. Extracts have also been prepared by

acceptor amino acid by sodium areenite using grinding with alumina according to McIlwainClostridium eporogenes suspension (34). The extracts were dialyzed against distilled

mmBmT" -ODIM -2+NHs+ !MON water and sodium sulphide at a temperature ofsvsmassAXSZZ - +A.02 USTA -5 to 0 C. As a rule dialysis of 4-5 hours is

pM~ xFxM Isufficient to depress considerably the endogenous50 aanine - M 47.9 1.3 respiration; longer dialysis causes a great loss in50 alanine 3 39 87 1.6 18 the enzymatic activity.Preliminary tests for enzyme activity were

50 alanine 20 45 1.6 57 made by observing the decolorization of MB or+ phenosafranin in the presence of substrate. After

100 proline dialysis of the enzyme no MB reduction was50 alanine 3 35.4 40.3 1.2 10 observed unless inorganic phosphate and di-

+ phosphopyridine nucleotide (DPN) were added100 proline too (32, 33). Both DPN and phosphate are

All the conditions are identical to those ex- ne for the oxidation of the substrate, aspressed in table 2. was confirmed by manometric experiments

(figure 4 and 5; table 4). The enzymatic activityshown that reactions i and ii re catalyzed by with TRIS (trihydroxymethyl-amino-methane)two different enzymes. Knowing these facts and or phosphate buffer, pH 8, without DPN is verykeeping in mind the amino acid donor-acceptor small; the residual activity probably arisingnature of coupled domination the reaction from incomplete removal of the DPN by dialysis.scheme I may be put forward. The data of figures 4 and 5 indicate that theFor the reaction, three enzyme systems are reaction is of the first order and that the oxida-

foreseen: (a) L-amino acid dehydrogenase tion does not proceed to completion, possiblysystem; (b) a-keto acid dehydrogenase system; attributable to the destruction of some cofactor.(c) amino acid reductase system of the hy- The enzyme system is active only on the -aminodrogenase type. In the scheme the amino acid acids. The rate of reduction falls in the order ofdonor undergoes oxidative demination by substrates given: alanine, leucine, norleucine,dehydrogenation through enzyme system I, and valine and isoleucine. Phosphate and DPN arethe a-keto acid formed is oxidatively decar- necessary for the oxidation of each of theseboxylated by enzyme system II. amino acids.These reactions occur simultaneously giving Aerobic oxidation also requires MB as an

as products 4 hydrogens, ammonia, carbon intermediary carrier similar to the analogousdioxide and fatty acid as shown in underlines, requirement by the glutamic, dehydrogenase ofThe transfer of the hydrogen proceeds anaerobi- von Euler (35, 36) and Dewan (37). With thecally to the third enzyme system (III) where the latter, MB replaces a flavin type carrier in theacceptor amino acid is consequently reduced. oxidation of glutamate (probably diaphorase);Aerobically the hydrogen transfer from enzyme presumably MB plays the same role in thesystems I and II proceeds through oxygen as oxidation of the aliphatic amino acids by extractsacceptor (IV). from obligate anaerobes. Cell suspensions, on the

L-AMINO ACID DEHYDROGENASE SYS other hand, have been shown not to require MBScheme I gives the reaction mechanism in the oxidation of amino acids (21).

pictures as a result of studies made with in*t Further confirmation of the results was

cells. More recently, studies have been carried furnished by spectrophotometric measurementsout in this laboratory with dialyzed cell-free at 340 mju of DPN reduction using the Beckmanextracts prepared by ultrasonic vibration (32, 33). spectrophotometer. Figure 6 shows the enzymaticThe present state of these studies, which are still reduction of DPN in presence of alanine; theunder way, will be considered in the following reduction is initially very rapid and quicklysections. reaches a maximum. The enzyme is specifically

Catalase was added before vibration to protect linked to DPN, TPN not being reduced under


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ml 02 A0p M/. 0A

Z.000 /

- Af vl~e A4

800 A

600 -25po/021}

400L

200 -

0 20 40 60 80 /00 20 /40TIME IN MINUTES

Figure S. Suppression of the physiological acceptor activity by sodium arsenite. The conditions arethose of table 3.

Curve A = 02 uptake in presence of alanine.A, = same + sodium arsenite 10-3 M.A2 = same as A1 + proline.As = same as A2 without arsenite.

R-CHNH2COOH (Donor) AEROBIOSIS

> ~ (2H) 1/202H20*, > s ~~~~~~~~~~~~~~(2H)

R-CNHCOOH

H2C - Carrier / ANAEROBIOSISsystem

R-COCOOH NH3 (2H) proline (Acceptor)

(Donor) Wi amino-valeric

H20 II (2H) i

glycine CH-3COOH +H3R C 2 (Acceptor

SCHEME I. Scheme for the Stickland reaction based on experiments with intact cells of Clostridiumsporogenes. End products underlined.

Reaction Ensyme system postulad INhibiorsI L-amino acid dehydrogenase KCNII a-keto acid dehydrogenase CHsICOOHIII Amino acid reductase AsA0,Nas


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TABLE 4 the same conditions. The DPN reduction isEffect of inorganic phosphate and of diphospho- proportional to the concentration of phosphate;pyridine nucleotide on the oxidation of L-alanine in the absence of phosphate a slow reduction

by Cloestridium sporogenes extracts does occur followed by a spontaneous reoxidationoxIMF= BOXWTS anew of reduced DPN (figure 6). As is noted in figure

ZxTJ __ _7 and table 4, DPN is reduced in the presence of+NH. -0, Q +N .-o2 ,2/ arsenate as well as phosphate, much lower

- concentrations of arsenate being necessary for theoM FaP same amount of reduction. Manometric experi-

1. Nondialyzed 16 16 0.5 15 34 1.1 ments on the oxidation of amino acids show the2. Dialyzed 225 4 4 0.5 9 18 1

same proportionality to the concentration of3. Same as 2 + 4 4.50.56 20 30 0.75 phosphate. The rate of aerobic oxidation is

m/7,5W maximal at pH 8. Ferricyanide replaces oxygenDPN as a hydrogen acceptor and suppresses the oxygen

4. Same as 3 + 13 21 0.8 - - - uptake under aerobic conditions.x/60 ar- Peroxide fomnation. The formation of hydrogensenate peroxide was investigated with extracts prepared

in the absence of catalase (table 5). Since in theEach Warburg flask contains 0.8 ml substrate

(M/20), 0.1 ml MB x/500, 1 ml buffer (pH 8, m/15); presence of these extracts the oxygen uptake wasin the side am, 0.5 ml extract (45 mg dry wt) of deprsed by catalae, cell-free extracts formC. 8porogenee; in center well 0.2 ml 20% KOH; hydoge peroxde which do not occur withtotal volume 3.2 ml. Enzyme and substrate were cell suspensions. The last hydrogen carrier in themixed at t = 0; gas phase, air; time, 120 min; extract system is MB which reacts as follows:temperature, 37 C. MBnd + 02 -0 MBo1 + H202. It is not known

C.-I~~~~~~~~~~~~~~~~

N /20.IVI6/ n+ ,13......1v

20 40 60 80 100120140 160TIME IN MINUTES

Figure 4. Effect of inorganic phosphate and DPN on the oxidation of .-alanine and valine. See table4 for conditions; phosphate in all; extract dialyzed 225 min; oxygen uptake is corrected for the blank.Curve I: oxidation of alanine + DPN; II: oxidation of valine + DPN; III: alanine - DPN; IV: valine- DPN.


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24 B. NISMAN (VOL. 18

300 )II 02O Iv Phosphate

DPN250

2001 110 ~ Phosphate

iso~ S150~~~

100 0w~=vV00000~~ Veronal

50~~~~~DPN

0

0 20 40 60 80 100

Time in minutesFigure 5. Effect of inorganic phosphate and DPN on the oxidation of -alanine. The conditions are

those of table 4; extract dialyzed 225 min. Curve I: oxidation of alanine in presence of phosphate andDPN; II: same as I without DPN; III: same as II with veronal replacing phosphate; IV: same as Iveronal replacing phosphate.

500 whether in the cell suspensions of C. sporogeneethe hydrogen carrer, which is replaced in the

I' cell-free extracts by MB, reacts with molecularE 400 / oxygen to form hydrogen peroxide; and if so,

whether there is present an enzyme stem otherIV) than catalase (38, 39) which breaks down theco 300 peroxide, or whether the final hydrogen carrier

reacts like the hydrogenase of Stephenson andAso a Stickland (28, 29): 2H2 .+ 02 -. 2H20.200 Reaction equilibrium. The oxidation of the

ctp 200- 6, / amino acids with DPN as electron acceptor is areversible reaction as shown in figure 8. DPNrdis completely reoxidized by the addition of

100 - a-keto acid and ammonia. Manometric experi-*vvt\ ments with alanine suggest a similar conclusion

since the addition of pyruvate completelyC 14 suppresses the oxidation of alanine, as measured1 2 3 4 5 6TIME IN MINUTES genee (15 mg) and M/15 phosphate; Ia: same with

Figure 6. Effect of phosphate concentration on M/30 phosphate buffer; II: same with 0.1 M TRISthe enzymatic reduction of DPN in presence of instead of phosphate buffer; III: same with 0.1 Malanine. Curve I: Reduction of M/7,500 DPN in veronal buffer. In all total volume was 3 ml andpresence of 0.2 ml extract of Clostridium spore- pH 8.


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200

0 150 . 0

4. . 41

100

0~~~~~~~~~~~~~450

I 2 3 4TIME IN MINUTES

Figure 7. Curve I: Reduction of M/7,500 DPN by u/200 alanine in presence of 0.2 ml extract of Clos-tridium sporogenes (15 mg) and of phosphate (M/20, pH 8), total volume 3 ml; II: same with x/60 arsenatereplacing phosphate; III: same as II, with x/300 arsenate.

TABLE 5 IHydrogen peroxide formation during the oxidation :I3O00

of alanine and glutamic acid by Clostridium Eeporognes extract not containing catalase 0

a8TlaT. -Ou +NHa ( 200\

Alanine 108 6.65 -1Na glutamate 182 9.5Na glutamate + catalase 116 | 10.7

x/15 phosphate buffer, pH 8; x/7,500 DPN; the 0 2 4 6 8 10 12 14 16other conditions are identical to those expressed TIME IN MINUTESin table 4. Figure 8. Curve I: Reduction of x/7,500 DPN

by 0.2 ml (15 mg) extract of Clostridium 8porogenesby ammonia formation. Moreover, chromate (dialyzed 240 min) in presence of 0.3 ml i/20graphicexeriments demonstrate the syn4thess alanine, 1 mls/15 phosphate buffer (pH 7); totalOf alanine from pyruvate and ammonia (40). volume 3 ml. II: Reoxidation of reduced DPN by

Phosphate in the oxidation of amino acids. The pyruvate and ammonia; 0.3 ml x/10 pyruvate andintervention of phosphate in amino acid oxidation 0.3 ml x/10 ammonium sulphate added as indicatedhas not been previously described. Lehninger by arrows.(41) and others showed that an esterification ofinorganic phosphate takes place in mitochondrial been clarified. With extracts prepared free fromsystems during the enzymatic reaction DPN,.d" acetyl phosphatase activity (40, 43) we haveDPNOX. In the cyclophorase system oxidizing found, starting from alanine, a considerableL-alanine, Still et al. (42) state that phosphate esterification of inorganic phosphate, usingdoes not participate in the amino acid oxidation adenosine-5-phosphoric acid as a phosphatereaction itself. In these two examples phosphate acceptor (table 6). The ratios, NH, liberated:acts at a secondary level. The mechanism of 02 taken up:P04 taken up, were 1:1:1.6.phosphate intervention in the reactions carried Experiments made in the absence of muscleout by the extracts of anaerobes has not yet adenylic acid showed no such esterification. As


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TABLE 6 TABLE 7Inorganic phosphate esterification during the oxida- Effect of the inhibitors on the oxidation of alanine

tion of alanine and pyruvate by Clostridium by Clostridium 8porogene8 extractssporogenes extract not decomposing acetyl phos- CONCENTRION IIBONphate

SUBSTRATE NADENYLIC +NH, |0 -0P1 +ACETIC Na azide M/300 0ACID ACIDNa arsenite 1/1,000 5

p w pf 5aM gAatoms pM pAM Na iodoacetate M/300 45Alanine, 40 0 11 22 0 K cyanide M/100 100Alanine, 40 40 11 22 16 - Hydroxylamine x/100 100Pyruvate, 40 40 - 30.3 23.5 35 Semicarbazide 1M/90 35

Conditions same as in table 4. M/15 phosphate 1m/15 phosphate buffer (pH 8); 11/7,500 DPN;buffer (pH 8), m/7,500 DPN, added to all sub- time, 60 min. All other conditions are identicalstrates. Experimental time, 60 min. to those expressed in table 4.

many respects the enzyme found in the liver bywill be mentioned later, the breakdown of the von Euler et al. (35, 46, 47) and Dewan (37) thata-keto acids by the extracts is phosphoroclastic of Escherichia coli (36) and that found in Neuro-(40, 43). One might therefore assume that the spora crassa (48). The clostridial enzyme differsaction of phosphate in this case is also secondary; in that it is specifically linked to DPN, whereasthe phosphate, facilitating the rapid breakdown the liver enzyme can be either DPN- or TPN-of a-keto acids, would shift the reaction equi- linked, and the E. coli enzyme is specificallylibrium towards deamination: TPN-linked.

alanine - o pyruvate + DPNH~d

-) acetyl phosphate + CO0

This assumption, however, is contradicted by The C. sporogenes glutamic dehydrogenase hassome of our experimental results: (a) some some properties in common with the otherextracts, not containing the a-keto dehy- L-amino acid dehydrogenase systems existing indrogenase, oxidize amino acids and yet require the same organism. For example, DPN cannot bephosphate; (b) monoiodoacetic acid and semi- replaced by TPN, and the reaction comes to ancarbazide, inhibitors of the dissimilation reaction equilibrium analogous to that described by vonof the a-keto acids, do not suppress the effect of Euler (35, 46, 47) and Dewan (37):phosphate on amino acid oxidation (table 7). Idglutamate + DPNOZ =An esterification of inorganic phosphate may also a-ketoglutarate + NH, + DPNdbe coupled to the enzymatic reduction of DPN. Also, the addition of a-ketoglutarate andThis problem is at present being investigated. ammonium ions causes complete reoxidation of

L-Glutamic dehydrogenase. Clostridium 8po- the reduced DPN; maximum activity is obtainedrogenes does not oxidize L-glutamic acid to any at pH 8. Aerobic oxidation of L-glutamic acidgreat extent; indeed, several workers have also requires addition of MB as an intermediatestated that it is a poor hydrogen donor (3, 4, 5, electron acceptor, and hydrogen peroxide is6, 9). We have found our extracts to contain an formed (as determined by the addition ofactive L-glutaxnic dehydrogenase (44). This catalase) with a consequent lowering of thediscrepancy can be explained by the fact that the oxygen uptake (table 5). Phosphate has only aslight catalytic effect on the aerobic oxidation ofassimilation of glutamic acid by gram positive L-glutamic acid. Glutamic acid dehydrogenaseorganis (45) requires an independent source of can be distinguished from the other aliphaticenergy, such as glucose or arginine. The glutamic amino acid oxidizing enzymes by its insensitivitydehydrogenase of C. sporogenes resembles in to KON and to hydroxylamine, both of which


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TABLE 8 appears to be about 10-15 times less active thanOxidation of glutamic acid by Clostridium the glutamic dehydrogenase system. However,

sporogenes extract when the oxidation is performed aerobically inthe Warburg in the presence of MB, the same

ADDIIONS+NHs0extracts show about the same level of activity

pm juM iaSems for both systems. A plausible explanation of thisVeronal 0 12 16.6 difference is that oxygen consumption underVeronal M/90 semicarbazide 16.6 16.6 aerobic conditions is limited by the carrierPhosphate 0 16 21.6 acting between DPN and MB.Phosphate m/90 semicarbazide 20.1 20.3 Transamination in teU oxidation of the aminoPhosphate m/90 KCN 20.3 21.6 acids. The existence in these bacteria of a very

Conditions as in table 4. m/15 phosphate buf- active glutamic dehydrogenase raises thefer (pH 8); M/7,500 DPN; time, 120 min. question of its actual physiological significance.

1.000 X

Sooo s __~_x

0~~~~~~~800~~, 1

'4 600i X0 /~~~~~~~~~

2600 /

2'400 26 8 10 12 14 16 1 20

TIME IN MINUTESFigure 9. Curve I: Reduction of M/7,500 DPN by 0.2 ml (5 mg) extract of Clostridium sporogenes

(dialyzed 240 min) in presence of 0.3 ml m/20 glutamate, 1 ml m/15 phosphate buffer (pH 7); total volume3 ml. II: Reoxidation of reduced DPN by a-ketoglutarate and ammonia; 0.3 ml x/10 ex-ketoglutarate( I ) and 0.3 ml M/10 ammonium sulphate ( I I ) added as indicated by arrows.

are strong inhibitors of the oxidation of the Braunstein (49) has postulated that it plays ana-amino monocarboxylic, acids. Cyanide, semi- important part in the oxidation of L-aminO acidscarbazide and hydroxylamine, by their inter- in the liver systems. Braunstein and Asarkhaction with the carbonyl group of the a-keto (50) and Nisman et al. (51) have shown thatacid, accelerate the rate of dehydrogenation of glutamic acid dehydrogenase does indeed playglutamate (measured in the Warburg micro- such a role in bacterial suspensions also. Inrespirometer or with the Beckman spectro- experiments with C. saccharobutyricum sus-photometer) by displacing the reaction equi- pensions (51) which do not carry out the SR itlibrium in favor of deamination (table 8, figure 9). was found that deamination of alanine ana-On the basis of DPN reduction measurements in erobically occurs only in the presence of a-keto-the Beckman, the alanine dehydrogenase system glutaric acid, according to this reaction:


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(transaminase) (dehydrogenase)CHCHNH2COOH + a-ketoglutarate

pCH.COCOOH + glutamatephosphate

DPN DPNH

/\[CO3COOH + C02 + H2] a-ketoglutarate + NH,

TABLE 9 TABLE 10Effect of a-ketoglutaric acid on the oxidation of Effect of a-ketoglutarate on the oxidation of alanine,

alanine by Clostridium sporogenes extracts saline and leucine by Clostridiumsaccharobutyricum extracts

GLUTARATE -0s +NHS__BST____a-KTo- -OS2 +NHa

PatS"=ATmGLUTARATENa glutamate none 11 11 Am ASIOmS AmAlanine none 26 16 Alanine none 0 0Alanine 5 30 18 Alanine 10 18.8 12.2Alanine 30 24 17 Valine none 0 0Valine none 24 14.3 Valine 10 15.6 11.3Valine 30 22 13.2 Leucine none 0 0

Leucine 10 7.6 6.1

m/15 phosphate buffer (pH 8); m/500 DPN; Glutamate none 22 16.6substrate, 30 ,&i; time, 90 min. All other condi-tions are identical to those expressed in table 4. m/15 phosphate buffer (pH 8); m/7,500 DPN;

substrate, 30 sm; time, 90 min. The other condi-An identical reaction starts the oxidation of tions are those expressed in table 4.alanine in cyclophorase preparations (42). WithC. sporogenes enzyme systems, we have ex- extracts is that KCN and hydroxylaminelperienced certain difficulties in determining reputed to form complexes with the carbony,whether an initial transamnation occurs previous groups of the a-keto acids and that of pyridoxalto the dehydrogenation reaction because: phosphate, inhibit the oxidation of the aliphatic

(i) The oxidation of alanine, valine, etc., when amino acids (see tables 7 and 8). Hydroxylamine,measured by the rate of MB reduction or by as shown by Cohen and Cohen-Bazire (un-oxygen uptake, is very rapid, being even faster published results), inhibits the SR with C.than that of glutamate. Sporogenes suspensions at a concentration of

(ii) The addition of a-ketoglutarate does not 10-' M. The inhibition of the DPN-linkedseem to increase the rate of the oxidation of ethanol dehydrogenase from liver and yeast byalanine to an appreciable extent (table 9). low concentrations of hydroxylamine has

(iii) C. sporogenes does not require vitamin recently been reported by Kaplan and CiottiBe for growth (52), and we have not been able to (54).prepare suspensions starved of this growth With group B bacteria which do not affectfactor. the SR (cf. p. 17) it has been easier to show

(iv) Under the experimental conditions used that the dehydrogenase system acts indirectly,some proteolysis occurs in the mixtures [about and that transamination with a-ketoglutarate isone jmol of glutamic acid being formed during the first step in the oxidation of amino acids.an experiment, as measured by the C. welchii Extracts prepared as before (32, 33) and dialyzeddecarboxylase technique (53)]. This may perhaps in the usual manner show no oxidation ofbe sufficient for the dehydrogenase system and alanine, valine and leucine. Addition of a-keto-so obviate the need of added a-ketoglutarate. glutarate restores the dehydrogenase activity in

(v) The only factor in favor of the occurrence these amino acids (table 10).of the initial transamination in these bacterial The addition of a-ketoglutarate, DPN, phos-


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phate and MB are necessary for the oxidation of a-keto acid and glutamate. The L-glutamic acidthese amino acids. DPN is known to take part formed is dehydrogenated through the corre-in the dehydrogenation of glutamic acid by sponding DPN linked dehydrogenase II formingC. saccharobutyricum (and very probably a-ketoglutarate, NH3 and DPNrd. The a-keto-phosphate also). The glutamic dehydrogenase of glutarate can be used again for the preminarythis bacterium has almost the same enzymatic trmination. Reduced DPN is oxidized toproperties as those of C. eporogens; it is activated DPN aerobically through MB with molecularby high concentrations of cyanide and hy- oxygen (and an intermediary diaphorase linkingdroxylamine. The addition of inorganic phosphate DPNd to MB (V)) and forming consequentlyfavors the phosphoroclastic breakdown of _ DPNW -* MB, + H101. Reduced DPN isa-ketoglutarate by shifting the reaction towards oxidized anaerobically through the action of adeamination. It should be remarked here that specific hydrogenase III catalyzing the reversiblethis enzyme functions anaerobically without any +2eadded hydrogen acceptor. The hydrogenase reaction 2H =± H2. The a-keto acid formed by

+. system I is metabolized oxidatively by systemscatalyzing the equilibrium reaction 2H = H2 IV, VI, VII with accompanying DPN reduction,seems to be linked to the glutamic dehydrogenase and the reduced DPN is reoxidized via theand enables the hydrogen transfer from donor to previously mentioned systems.acceptor (unpublished results).Scheme II shows that the dehydrogenation

oeaction involves a preliminary tranamination I Another important system in the coupledrf the r-amino acid with a-ketoglutarate to the deamination is the a-keto acid dehydrogenase

R CHNH2COOH L-glutamate

I ~~~~~~IIlffl3

R-CQ-COOH C-ketoglutarate

znK(2H) AEROBIOSIS

CoA DCPNC2(2H) D; a B ;\PO4- DP~~~~~~~red(X~~red 2F-~~~~~ VXox H20R-CO-/O-P0oxDN

AMP ANAEROBIOSISADP

VIIfADP

R-COOH bATP

ScAdis II. Dehydrogenation reactions in the oxidation of Lamino acids by extracts of clostridianot carrying out the Stickland reaction (Clotridium saccharobutyricum). Final products underlined.RZacien ESsyM CefacsrsI Transaminase Pyridoxal phosphateII L-glutamic dehydrogenase DPNIII Hydrogenase Not knownIV a-keto acid oxidase DPN, CoA, PO,, DPT, a-lipoie acidV Diaphorase? FADVI Transacetylase P04VII Labile phosphate acceptor system CoA?


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30 B. NISMAN (VOL. 18

system; suspensions of strict anaerobes have plays the part of an intermediary electronbeen shown to carry out the following anaerobic acceptor. A system similar to that found inbreakdown of pyruvate: C. saccharobutyricum which decarboxylates0130OOH0 H+20--CHsCO0H +002+1H2. pyruvate oxidatively to yield acetyl phosphate,

C02 and molecular hydrogen has been extractedUnder aerobic conditions it was shown that from suspensions of Neisseria caproica by Peelclostridial suspensions decarboxylate pyruvate and Elsden (private communication).and other a-keto acids oxidatively (21, 23a) Importance of inorganic phosphate. Theaccording to the reaction: requirement for high inorganic phosphateR-COCOH + X 02 --R-RCOOH + C02. concentrations for pyruvate oxidation, asdescribed for C. butylicum (55, 56), holds also for

By freezing and thawing suspensions of Clostri- extracts obtained from C. porogenes and C.dium butylicum, Koepsell and Johnson (55) and saccharobutyricum (32, 40, 43, 57). Figure 10Koepsell, Johnson and Meek (56) prepared cell- shows the effect of phosphate concentration onfree extracts that oxidatively decarboxylated pyruvate dissimilation. It can be seen that highpyruvate, but high concentrations of inorganic concentrations are necessary both for optimalphosphate were found to be necessary (5 equiva- oxidative activity (oxygen uptake) and acetyllents of phosphate to 1 of pyruvate). Acetyl phosphate formation, and that an obviousphosphate was formed from the pyruvate, but proportionality exists between the oxygenMB was not reduced under any conditions. The uptake and acetyl phosphate formation. It mustauthors supposed this to be due to the absence be mentioned that some pyruvate is dissimilatedof the necessary hydrogenase. The degradation of nonoxidatively by C. sporogenes and C. 8accharo-pyruvate and other a-keto acids with bacterial butyricum extracts in the absence of phosphate.suspensions has already been discussed (see page The requirement for such high concentrations18). With cell-free extracts obtained by ultra- of phosphate is not characteristic of othersonic vibration (32, 40, 43) it has been found bacterial pyruvate oxidizing enzymes. Stumpfthat the degradation of pyruvate can be ac- (58) presented evidence that the pyruvatecounted for by the following set of reactions: oxidase of Proteus vulgaris does not require

Aerobiosi-s phosphate either aerobically or anaerobicallyAob_ s with ferricyanide as electron acceptor. MoyedCHSCOCOOH - a-- and O'Kane (59) have fractionated the pyruvate

CHCOO-PO&H2 + CO + H20 oxidase system of P. vularis and found that twoammonium sulphate fractions must be combined

AnasrObiosis to obtain pyruvate oxidation. CoA is not requiredCHsCOCOOH -PO' in this system, but in its presence sulphanilamide

CH1COO-POHE + C02 + H2 can be acetylated. Ochoa et al. (60, 61, 62)demonstrated that in the pyruvate oxidation(C. saccharobutyicum) system of E. coli phosphate was required only for

CHCOCOOH - I--) acetyl phosphate formation, i.e., was bound toCH,0OO-POsH2 + C00 + 2H + 2 the activity of Stadtman's transacetylase

system (63, 64). They found that in the presence(C. sporogenes) of another enzyme capable of detaching activeMolecular hydrogen is liberated only by C. acetate and condensing it with oxalacetic acid tosaccharobutyricum; C. sporogenes requires ferrn- make citrate inorganic phosphate becamecyanide, oxygen, or a physiological electron unnecessary. Another bacterial pyruvate oxi-acceptor system (glycine or proline reductases as dative enzyme, similar to that found in thewell as reductive amination by alanine synthase) E. coli extracts described by the previouslyfor the oxidative decarboxylation, whereas mentioned authors, is found in StreptococcusC. saccharobutyricum does not. The latter faecalis (65). This enzyme system requires CoAorganis needs instead a system which can and phosphate for pyruvate dehydrogenation as

+2e measured by dismutation and acetyl phosphatecatalyze the reaction 2H = H2 and so probably-20 accumulation. The initial cell extracts appear to


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14

12/

U'

0.

0.

-j

I-

4

20

0 M/450 M/225 M/12 /55 M/28 M/14PHOSPHATE CONCENTRATION

Figure 10. Effect of phosphate concentration on the oxidation of pyruvate and acetyl phosphateformation; 50 PM pyruvate + varying concentrations of phosphate + 0.5 ml Clostridium sporogens(40 mg dry wt) extract (not splitting acetyl phosphate). Final volume 3 ml. Gaseous phase, air. Tempera-ture 37 C. Experimental time 40 min. Curve I: Acetyl phosphate formation; II: oxygen uptake.

contain a hydrolytic enzyme since less acetyl acts as an electron acceptor in both aerobic andphosphate accumulates than expected, but on anaerobic reactions. Among the inhibitorspurification of the enzyme there is a rise in the tested, the system has been found to be sensitiveaccumulation of acetylphosphate per mole of to iodoacetate (M/500) and high concentrationspyruvate oxidized. The need for high concentra- of azide (M/90). Dinitrophenol (M/1,500) andtions of mineral phosphate in the a-keto acid arsenite (M/900) do not inhibit the oxidativedehydrogenase systems of clostridia can be reaction. Phosphate is replaceable by arsenate,explained essentially by the presence of trans- lower concentrations of the latter sufficing toacetylase which catalyzes the reversible reaction give the same rate of reaction. Methylene blue,demonstrated by Stadtman (63, 64) although reduced by the system, is not necessary

for the oxidation if the extract has not beenAcetyl-CoA ' Acetylphosphate dialyzed too long. After dialysis exceeding 6

CoA hours the oxidation of the a-keto acids is stronglySince the nonoxidative reaction of pyruvate is enhanced by MB.competing for pyruvate, it becomes under- Fomation of hydrogen peroxide. The formationstandable that high concentrations of phosphate of peroxide could not be demonstrated in thewould influence the reaction in the oxidative aerobic oxidation of pyruvate. Addition ofsense. catalase did not influence the oxygen uptake, butThe optimal pH for oxidative dissimilation of the possibility of peroxide formation is not

pyruvate by extracts from C. sporogenes and eliminated since the substrate is able to reactC. saccharobutyricum is about 6.5. Ferricyanide nonenzymatically with hydrogen peroxide (66,


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67). One part of the pyruvate is oxidized to as well as for the pyruvate oxidase from the higheracetic acid, carbon dioxide and hydrogen organism (77, 78, 79, 80, 81).peroxide, and the latter then oxidizes the The pyruvate oxidase systems of higherremaining pyruvate to acetic acid and carbon organ can be represented as follows:dioxide nonenzymatically. The over-all reaction at Igives the impression that no hydrogen peroxide P vahas been formed, but we are inclined to believe acetyl-enzyme It acetate + enzymethat this system does form peroxide during the M4CoAreaction (68, 69). a

Cofactors involved in pyruate oxation in C. acetyl-CoA - acetate + enzymesporogenes extracts. Recently two important IV4SAMcofactors, conting sulfhydryl groups, a-lipoic acetyl-SAM (SAM - sulfonamide)acid (70, 71) and coenzyme A (57, 72, 72a),have been found to be involved in pyruvate Reactions I and II are catalyzed by purifiedoxidation systems. Lynen et al. (73, 74) suggested oxidase systems with dyes or oxygen as electronthat the oxidation of pyruvate (or of acetalde- acceptors. The existence of a high energy inter-hyde-like compounds derived from pyruvate) by mediate in pyruvate oxidation can be shown bydehydrogenase might occur through combination the fact that acetyl-sulfonamides or acetylwith the SH-groups of CoA, the resultant phosphate (77, 80) is formed in the absence ofcompound giving rise to acetyl-CoA and carbon ATP, when appropriate acceptor systems anddioxide by subsequent oxidative decarboxylation. CoA are added. This primary acetyl-intermediateThese authors have isolated and characterized could be an acetyl-enzyme complex as shown inacetyl-CoA and have found it to be identical the scheme. Since acetate is formed in thewith active acetate (73, 74). The pyruvate absence of CoA and DPN (when dyes or oxygendehydrogenase would accordingly act as follows are used as electron acceptor), the identity of the(73, 74): first formed compound with acetyl-enzyme

0 OH 011 I 11Al

CE6CCOOH + R-SH CHECCOOH - * CEHC + CO2l lSR SR

0 OH 011 I H

CH.CH + R-SH-- CH.C CHECl ISR BR

(R-SH - CoA)

The mechanism proposed for the function of complex as shown in the scheme is most likely.glutathione in the glyoxalase system (75, 76) This postulated intermediate might also exist inand in the oxidation of glyceraldehyde by the bacterial systems where CoA apparently playstriose phosphate dehydrogenase system are no part in pyruvate oxidationi.e., P. vulgarissomewhat similar (76). Nisman and Mager (57) and also the protozoan Tetrahymena pyriformis.and Korkes et al. (72, 77) as well as Littlefield The specific intermediate may be postulated asand Sanadi (78) agree with Lynen and Reichert's an acetyl-POF (POF - pyruvate oxidizingproposed mechanism. Nisman and his associate factor) enzyme complex with the acetyl linked(40) have shown that the three cofactors, CoA, to sulfur as in acetyl-CoA. According toDPN and cocarboxylase, are required for Schweet and Cheslock (80) the first stages ofpyruvate oxidation by extracts obtained from pyruvate oxidation are the same with fern-C. sporogenes. The same cofactors are required cyanide, dyes, oxygen as with DPN and CoAin the pyruvate oxidation systems of E. coli (80). The direct reduction of DPN catalyzed by(72), S. faecalis (65) and C. saccharobutyricum purified pyruvate oxidase from pigeon breast


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1954] THE STICELAND REACTION 33

TABLE 11 TABLE 12Comparative oxidation of a-keto acids by Dependence of the oxidation of Na pyruvate on the

Clostridium sporogenes extracts presence ofcoenzyme A and inorganic phosphateaccumulating acyl phosphates by Clostridium saccharobutyricum extracts

SUBSTATE -°AC0YL P04 -O,/ACL -0BF71MW P0, TRIS muirix PwosP2ATZ BUTTERVATE

gsaemspM ADDiTION _ _T

Pyruvate 23 12 0.96 aceto. ace&to- ho"a-Keto valine" 18 9 1 -Pv -0 ace- _O a ep-t"'a-Keto leucine" 8 4 1 ~ aeer)

Each Warburg flask contains the substrate + PM PM JM s

phosphate buffer K/15, pH 6.6. In the side arm 0 0 1 0 0 2 00.5 ml dialyzed extract (40-45 mg dry wt); finalvolume3 ml. In the centerwell 0.2ml20%oKOH. CoA 0 1 0 37.8 36 9 0.47Enzyme added to the substrate (50 pM) at 0 6 U/mltime; experimental time, 40 min; temperature,37 C. CoA - 42.2 38 10 0.45

12 U/mlmuscle with the consequent formation in presenceof CoA of acetyl-CoA was demonstrated by Each Warburg flask contained 0.5 ml substrateLittlefield and Sanadi (78), showing that reaction (0.1 x) + 1 ml phosphate buffer pH 6.5, (0.2 K)

,isaprothpodtsem or TRIS buffer (0.1 K) pH 7. In the side bulb 0.5III tisalonpartiof ith pyruvatereoxidatio noyten ml enzyme (50 mg dry wt). Enzyme and substrateIn this connection it is interesting to note that were mixed at t - 0. In the center well was 0.2Seaman (81) has demonstrated that a-lipoic ml 209% KOH. Total volume 3.2 ml; experimentalacid (POF) is required for the acyl-transfer but time, 120 min; temperature, 37 C; gas phase, air.not for oxidative activity as measured by 2-6chlorophenol-indophenol reduction in the Py e dehydrogenase system of C. saccharopyruvate dehydrogenation of Tdrahymena pyri- butyricm. The system shows the same propertiesformis enzyme. as that of C. sporogenes with respect to its phos-

Comparison of the dehydrogenase activity on phate requirement and sensitivity to inhibitors.different a-keto acids. The pyruvate dehy- Nisman and Mager ($7) have found that whendrogenase system of C. sporogenes oxidizes other extracts of C. arobutyricum are prepard bya-keto acids in addition to pyruvate (40). in ultrasonic disintegration without the addition ofextracts not showing acetyl phosphatase activity, catalae (or a reducing compound), CoA issuch oxidations resulted in accumulation of acyl required, and is effective only in the presence ofphosphates corresponding to the a-keto acids inorganic phosphate as is shown in table 12. Noinvolved [determined by the hydroxamic acid acetyl phosphate could be detected by thetest of Lipmann and Tuttle (82)]. The following method of Lipmann and Tuttle (82), and theacyl phosphates have been found (table 11): extract did not destroy synthetic acetyl phos-

phate, indicating the absence of an acetylPyruvrate -~acetyl phosphate phosphatase. The acetyl-bound compound formed

a-Keto ,P-methylbutyrate _isobutyryl phosphatea-Keto i-methylvalerate iisovaleryl phosphate In the oidative reaction was removed in this

case through a condensing mechanim formingThese acyl phosphates are the energy rich acetoacetate, analogous to that described byintermediates corresponding to the fatty acids Chou, Novelli, Stadtman and Lipmann (83)formed either in the SR or in the oxidation of and Stadtman (84). In fact, significant amountsamino acids or a-keto acids when oxygen is the of acetoacetate (estimated as acetone) have beenelectron acceptor. Paper chromatography (40) found to be formed by this system (table 13).on the same experimental mixtures as those Under anaerobic conditions, only smallused for the acyl phosphates demonstrated the amounts of the hydrogen liberated in thesame fatty acids as those formed in the corre- oxidative decarboxylation of pyruvate were usedsponding SR. for the reduction of the Ca compound formed in


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TABLE 13 against pyrophosphate + potassium chloride,Effect of phosphate and CoA on release of CO, and they require in addition cocarboxylase and DPN

H2 from pyruvate by Clostridium for the oxidation of pyruvate. Some extractssaccharabutyricum extracts have been found to accumulate acetyl phosphate;--- - - others, as previously mentioned, do not ac-

BEUY= ADDITION tu +H2 +COs R cumulate it, nor do they decompose addedVATE.M- ____ - acetyl phosphate. This would not be surprisingA M AW were the former extracts dependent on phosphate

TRIS CoA (6 U/ml) - - - for the pyruvate oxidation and the latter not,TRIS none - - but in fact, both types of preparations have beenPhosphate none 2 1.5 2 0.75 found to require inorganic phosphate (40, 43,Phosphate CoA (6IU/n)16 11 13 0.84 57). From scheme III one can see that the

Each Warburg flask contains 15 mm Na dehydrogenation stem I requires eitherpyruvate + 1 ml phosphate buffer 0.2 m, pH 6.5 transacetylase (II) (63, 64) or any other suitableor TRIS buffer 0.1 x, pH 7. In one side arm 0.5 acetyl-acceptor system capable of liberating theml enzyme (40 mg); in the second side arm, 0.2 CoA (III). Where transacetylase (II) is m gml 7.6 N sulphuric acid for stopping the reaction; (extracts not accumulating nor decomposingin the center well 0.2 ml 20%9 KOH in the flaskused for hydrogen estimation, and nothing in the acety l phosphate istiaflask used for CO, + H, output. Final volume requirement of mineral phosphate in the oxda-3.2 ml; enzyme and substrate were mixed at 0 tion of pyruvate, one may presume that thetime; experimental time, 90 min; gas phase, N2. phosphate intervenes at some other point of the

Acetyl phosphate or acyl phosphate

gP 4 1| PhosphotransacetylaseDehydrogenase

Pyruvate ) Acetyl-CoA + CO2 + DPNH + H+or2

(another sC-keto acid) ((Condensing enzyme

Acetoacetate (Acetoacetyl- CoA)Scummn III. Acetyl-acceptor systems involved in the dehydrogenation of pyruvate by Clostridium

saccharobutyricum extracts.

the reaction. This can be seen from the fact that enzymatic system. In a private communicationthe ratio of hydrogen liberated to carbon dioxide Dr. Lynen suggested that this general require-evolved is close to unity (table 13). The complete ment of inorganic phosphate in the pyruvatelack of activity of the enzyme system in the oxidation could be explained by assuming that itabsence of phosphate and CoA has led us to still intervenes in acetyl phosphate formation,conclude that CoA is directly involved in the but that the presence in the extracts of a phos-major reaction of pyruvate dehydrogenation (see phate acceptor system would result in a finalscheme III). It is interesting that extracts of negative hydroxamic acid test. Such a possibilityC. 8accharobutyricum condense active acetyl to cannot be elminated.acetoacetate. Since this organism reduces Influence of ATP on the pyruate oxidation.acetoacetate to butyrate in the presence of Some C. saccharobutyricum extracts not splittingmolecular hydrogen (85, 86); this may be taken acetyl phosphate (40) have shown rather un-to mean that acetoacetate, or a compound in expected ATP requirement for pyruvate oxida-close equilibrium with it, such as acetoacetyl- tion (87). Under aerobic conditions adenosine-5-CoA, is an intermediate in butyrate synthesis by phosphate (AMP) could replace ATP, but therethis organism (86a). was a certain lag period suggesting a preliminaryWhen such CoA requiring extracts are dialyzed synthesis of ATP by some secondary reaction.


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TABLE 14 TABLE 15Effect of ATP on the anaerobic dissimilation of Effect of ATP concentration on the anaerobic

pyruvate by Clostridium saccharobutyricum breakdown of pyruvate by extracts ofextracts not splitting acetyl phosphate Clostridium saccharobutyricum

NON- NY-ADDITIONS H2 .p~j +AaZ. ONI- Al? -PTRU- +H., +ACE- pDROX- PidH2zDMMONS ; sVOLv- VATE TIC DATrVE VATE TATE AE_ED ACI0D REAC-C.

TI1ON _ _ _ _ _ _ _

pM sm PM __ Phosphate buffer0 0 8.9 2.5 6.4 PM pM PM M JAM AMATP (3pm) 2.4 12 6 6 13.3 - 12 11.2 21 0.2 1.7ATP (6 pm) 9 30 11 10 26.6 28.7 18 19 36.7 1.2 2.0ATP (10 #) 15 30 17 13 26.6* 36.6 26.6 28 36 0.9 1.4CoA (15 Lipmann 0.5 11 6.5 4.5 39.9 34.4 35 69 0.2 2.0

unitsAMP (12.5 pm) 0.5 16.5 3.5 13 TRIS bufferDPN (1 m) 0 8.9 2.5 6.4DPT (100pg) 0 8.9 2.5 6.4 13.3 - 6 6 23 0.8 3.8a-Lipoic acid (30 0 8 3 5 26.6 20.7 11.5 13 49 0.6 4.3

units) 39.9 37 26.8 30 79.7 - 3.053.2 33 30 - 110 0.7 3.6

Substrate, 30 px; other experimental conditions - - I _IIare those described in table 13. * +20 units CoA.

* Pyruvate -. acetate. 50 pM of Na pyruvate + 11 ml TRIS buffer0.2 m, pH 7.2 (or 150 om phosphate pH 6.8) +

How exactly this ATP acts is difficult to envisage, 4 piM MgSO4 + 80 pm KCl + 0.5 ml bacterialbut it was thought that perhaps it might be extract (35mg dry weight dialyzed 5 hours againstinvolved in some cofactor synthesis. Were ATP 0.3% KCl + 0.1% Nags). In the center well 0.2active in cofactor synthesis then the addition of ml 20%o KOH, final volume 3.2 ml. Enzyme andthe .inquesionhsubstrate were mixed at 0 time, experimentalthe cofactor n question should stimulate the time 240 min. In the second side bulb: 0.2 ml

system in absence of ATP. Such stimulation i 5 N H2,04 for stopping the reaction, gaseousnot observed when FAD, DPN, CoA, DPT and phase, Na; temperature, 37 0; substrate, 50 pM.thioctic acid are added, either singly or incombination (table 14). Another possible explana- TABLE 16tion is that ATP might be involved in a system Lithium acetyl phosphate dissimilation byother than phosphotransacetylase or the con- Clostridium saccharobutyricum extract indensing enzyme system, which removes the presence of ATP and adenylic acidactive acetate from the CoA complex. WlCBTION AMMONS -ACETYLRecent experiments carred out by Wiesan- TIE EOSIATE

danger and Nisman (87) with extracts of C. min jam

saccharobutyrimum have shown that the oxidative 0 0 0activity (measured by hydrogen evolution) is 30 0 1.2proportional to the concentration of ATP. High 30 AMP (12.5 pM) 5.3concentrations of ATP have been found to cause 30 ATP (5 Am) 5.1an initial lag phase of 60-90 min in the hydrogen 30 CoA (20 Lipmann units) 1.9evolution. The pyruvate oxidation system 7 pm of lithium acetyl phosphate were incu-operates in the absence of phosphate buffer bated in narrow open tubes with 30 mg extract +needing, however, higher concentrations of ATP 1 ml m/15 phosphate buffer, pH 6.5; final volume(table 15). Some of the extracts used showed an 3.0 ml.additional requirement of CoA. There wasneither ATPase activity nor transacetylase almost completely recovered as acetyl hy-activity in these extracts. droxamic acid, the dialysis probably not beingSome ATP (about 15%) is split initially in sufficiently exhaustive to remove all acetate

presence of the enzyme system along which is present in the extract. The estimation of in-


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AMP - ADP IV

Acetyl phosphat Acetate(Acyl) M red 02

I 4|ADP ATP r

I-~~~~~~ICH3C0C00He-11poXcads X DP Acetyl-CoA + DPNH + H + C02

or CoA w00R-COCOOH V)III (C. saccharobutyricum

ATP eAMP CoA C4-Com und Xred

____ L~~ ~~~~~~~~~~~X)x H2C. saccharobutyricum (acyl phosphate) ox 2

glycine reductaseAcetate proline reductase V

amino acid synthaseC. sporogenes

ScHEWm IV. a-Keto acid dehydrogenation by Clostridium saccharobutyricum and C. sporogenes(acetyl-acceptor and donor systems).

(a) System I represents the two isolated fractions necessary for pyruvate oxidation in bacteria,where the CoA and DPN are involved in the oxidative decarboxylation and electron transfer.(Escherickia coli, Streptococcus faecalis, C. sporogenes and C. saccharobutyricum.) Pyruvate isinitially transformed (in presence of thioctic acid - POF, and DPT) into acetaldehyde-like com-pound X which presumably appears to be identical in all pyruvic oxidases. This intermediate isthen transferred, in the above mentioned systems, to the CoA and DPN fraction and conse-quently decarboxylated into Acetyl-CoA + C00 + DPNod. However, in the higher organismsystems, as well as in Tetrahymena pyriformis or Proteus vulgaris, the compound X is susceptibleto be transformed into acetate in absence of CoA and DPN.

(b) Enzyme system II is phosphotransacetylase catalyzing: Acetyl-CoA Gus Acetyl phosphate +CoA (C. saccharobutyricum; C. sporogenes).

(c) Condensing enzyme III liberates the CoA from the complex and forms acetoacetate (C. sac-charobutyricum).

(d) Enzyme system IV = labile phosphate acceptor system, which decomposes acetyl phosphate toacetate + energy rich bond (C. saccharobutyricum; C. sporogenes).

(e) Enzyme system VIII liberates the CoA in presence of ATP and forms presumably: Phosphoryl-CoA + AMP + Acetyl phosphate (C. saccharobutyricum).

(f) DPNd formed in the reaction can be reoxidized by VI - Hydrogenase, with subsequent evolu-tion of H2 (C. saccharobutyricum).

(g) DPN,.d can also be reoxidized by: glycine reductase, proline reductase, or the amino acid syn-thases, V (C. sporogenes).

(h) Aerobically (C. sporogenes and C. saccharobutyricum) DPNd is reoxidized through the MBsystem (probably diaphorase) VII.

organic phosphate (Pi) liberated from the ATP Schweet and Cheslock (80) demonstrated theduring pyruvate oxidation shows that two formation of a high energy intermediate in themolecules of Pi are liberated per molecule of early stages of the pyruvate oxidation (acetylATP when the reaction occurs in TRIS (tri- phosphate or acetyl sulphonamide formed in thehydroxymethyl-amino-methane) buffer (table absence of ATP, when appropriate acetyl15) or in a phosphate buffer. Lipmann (88) found acceptor systems + CoA are added). Thethe acetate activating system that he investigated splitting of ATP during pyruvate oxidationsplit ATP to pyrophosphate + AMP, and reaction to orthophosphate and AMP suggests


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19541 THE STIC ND REACTION 37

strongly that in the C. saccharobutyricum enzyme (89). A more detailed study of this enzymicsystem ATP might be involved in the. preliminary system as correlated to phosphorylation reactionsactivation of the CoA to phosphorylICoA which is now underway. The enzymic systems involvedwould be nee to this specific acetyl acceptor in the Stickland reaction are all DPN-linked.system. Further work is necesy to solve this The DPN can be in a loosely bound form, as inproblem. It should be mentioned that ATP and the case of the L-amino acid dehydrogenaseAMP activate the splitting of Li acetyl phosphate system, or in a more firmly bound form, as in theby the same extract as shown in table 16. The case of the a-keto acid dehydrogenase system.over-all scheme of pyruvate dehydrogenation The nature of the other intermediary hydrogenthen becomes that shown in scheme IV. carriers is unknown.

Another point which should be mentioned isTHE AMNO ACID REDUCING SYSTEM that other oxidation-reduction reactions may be

The most characteristic enzyme system in the considered as unspecific SR reactions. Her themetabolism of obligate anerobes is that reducing hydrogen donor instead of being an amino acidthe acceptor amino acids. This system, as has can be ethanol, glucose or some other substrate.already been pointed out, is present only in the Ethanol dehydrogenation with these extracts

Log'. ''f200-_i

150 - S.W°1500

2 4 6 8 to 12 14TIME IN MINUTES

Figure 11. Curve I: Reduction of DPN (X/7,500) by 0.2 ml (15 mg) extract of Cloetridium sporogenes(dialyzed 240 min) in presence of 0.3 ml alanine (M/20), 1 ml phosphate buffer (m/i5, pH 7). Total volume3 ml. II: Reoxidation of reduced DPN by 0.3 ml (u/l0) glycine (I ). III: Reoxidation of reduced DPNby 0.3 ml (X/10) proline (11).

proteolytic subgroup (A.P.4) of clostridia which occurs also through DPN linked enzyme systemscarry out the Stickland reaction. The discussion (89). Glucose dehydrogenation by the glycolyticin this section will be brief as there are little enzymic system also needs DPN (89). One mayexperimental data (89). Extracts which contain conclude therefore that DPN linked dehy-the reducing enzyme system can be obtained by drogenase systems play the role of hydrogendisintegrating bacterial suspensions in a hydrogen donors in this particular type of oxdo-reductionatmosphere (89). Such extracts take up hydrogen reactions.in presence of the acceptor amino acids. The same It should be added that there is some evidenceextracts have been found to carry the classical that two energy rich bonds are formed during theSR. Stickland reaction (see table 6). If we consider

Effect of DPN on the hydrogen uptake. Figure the formation of a single energy rich bond like11 shows that DPN is also involved in the acetyl phosphate, we have: -AF - -16,000amino acid reduction. When DPN is enzy- cal. This will indicate that the coupled deamina-matically reduced by alanine, the addition of tion reaction is a potential source of energy richglycine or proline reoxidizes the reduced DPN; compounds for cellular metabolism. Further workthe uptake of molecular hydrogen by glycine or is necessary to clarify the nature of the hydrogenproline is also increased in the presence of DPN carriers and to determine the free energy released


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R-CHNH2COOH (D) AEROBIOSIS

ADP M~~~~red~~ 02(H, I~AP

20

HO(NH3 ;5 4 H 0

IVR-COCOOH (DI')

(2H) DPNOX

CoA)\R-CO- CoA ANAEROBIOSIS

\_5/r ~_° - (2i;81 aa H2 Vll

R-CO-O-PO3)ADP

4 ATP (Acceptor amino acid

24: glycine or proline)AMP m (A)

/9ADP Reduced productsR-COOH

SCaMd VRctien Enyme CefvcorsI L-amno acid dehydrogenase DPN, P04II a-keto acid dehydrogenase DPN, DPT, CoA, a-lipoio acid, P04III Amino acid reductase DPNIV Diaphorase? FADV Transacetylase P04VI Labile phosphate acceptor system CoA?VII Hydrogenase 7(a) The donor amino acid D is oxidatively deaminated to a-keto acid (D'), ammonia and DPNHS

(reduced DPN) by the reversible reaction catalyzed by enzyme system I.(b) D' undergoes oxidative decarboxylation to CO, Acetyl-CoA and DPNH2 by the action of enzyme

system II.(c) DPNH, formed during the reactions catalyzed by enzyme system I and II is transferred to the

reducing amino acid acceptor system III where the acceptor A is reduced according to the re-action:

2H + X = XH,

or DPNH2 + X = DPN + XH2

(d) Aerobically DPNH2 is reoxidized by MB which becomes MBH,. MBH2 in turn is reoxidized bymolecular oxygen to MB + H,02.

(e) The reversible reaction I where DPN is reduced to DPNH2 may be coupled to an inorganic phos-phate esterification (ADP -- ATP).

(f) The acetyl-active is removed from the CoA by transacetylase and transformed to an acyl phos-phate (V).

(g) Acyl phosphate is decomposed by a labile phosphate acceptor system (AMP -* ADP) or (ADPATP) to a fatty acid and an energy rich bond (VI).

(h) Enzyme system VII is a hydrogenase which activates molecular hydrogen.(i) The intermediary carrier system IV acts between DPN.d and MB and is supposed to be a di-

aphorase (FAD enzyme).


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during the reaction. Scheme V summarizes the teolytiques du groupe de Cl. sporogenes.sequence of the Stickland reaction as it now Formation par reaction de Stickland desappears from the studies with cell-free extracts. acides isobutyrique, isovalerianique, et

val6rianique optiquement actif. Ann. inst.Note added in proof: Experiments on the Stick- Pasteur, 75, 291-304.

land reaction by Mamelak and Quastel (90) have 14. WOODs, D. D., AND TRIM, R. 1942 Theconfirmed our results. metabolism of amino acids by Cl. welchii.

Biochem. J., 36, 501-507.REFERENCES 15. PRhVOT, A. R. 1948 Manuel de determina-

tion et de classification des bactdries anadro-1. KnEES, H. A. 194 The L and n-amino acid be.MsoPrs rneoxidases. Biochemical symposium, no. 1 16. CW&nON, B. P. AND BraRe H. A. 1947Cambridge University Press. *6C*ON .PADB tH .14

CamK~zbridge A. 1951UniversityPress.no Amino-acid fermentations by Cl. propioni-2.KisH.In The Oxidtonof,pamin1 pcum and Diplococcus glycinophilus. Arch.acids. In The enzymes, vaol. II, part 1, pp. Biochem., 12, 165-180.

499-535. Edited by Sumner, J. B., and Bohm,1,1510499-535. Editedeby umer, JeB.,a 17. EL-SHAZLY, K. 1952 Degradation of proteinN Yb in the rumen of the sheep. 1. Some volatileN. Y fatty acids, including branched chain3 . H 1 isomers, found in sivo. Biochem. J., U,tions by which Cl. 8porogene8 obtains its 640-647.energy. Biochem. J., 28, 1746-1759. 18. EL-SHAZLY, K. 1952 Degradation of protein

4. STICKLAND, L. H. 1935 The reduction of in rumen of the sheep. 2. The action ofproline by Cl. sporogenes. Biochem. J rumen micro-organism on amino-acids.29, 288-290. Biochem. J., 1, 647-653.5. STICKLAND, L. H. 1935 The oxidation of 19. SmssAc, R., RAYNAUD, M., AND COHEN, G. N.alanine by Cl. sporogenes. Biochem. J., 1948 VaPation du type fermentaire des29, 889-*896. 194 rian dut rmnae des

6. STICKLAND, L. H. 1935 The reduction of bactdries ana6robies du groupe de CZ.8porofgene sous l'influence du glucose.glycine by Cl. sporogenes. Biochem. J., Ann. inst. Pasteur, 75, 305-39.

29,896-898. 20~~S. JAME, A. T., AND MARTN, A. J. P. 19527. WOODs, D. D. 1936 Further experiments on

the coupled reactions between pairs of amino Gas-liquid partition chromatography: theacids induced by Cl. 8porogene& Biochem. separation and micro-estimation of volatileJ.,,,30 ,,1934,196* fatty acids from formic acid to dodecanoicJ., 30, 193419u. acid. Biochem. 3., 50, 678-60.

8. NISMAN, B., RAYNAUD, M., AND COHEN, G. N. 21. NISAcN, B. AND VINET, G 1949 Le catabo-1948 Extension of the Stickland reaction li.smex'oxyD des acides 19n9 ce lesto several bacterial species. Arch. Bio- bie anafobes sties Ann. inst.chem., 16, 473-474. bacteuresana7robies stctes. Ann. inst.

9. KOCHOLATY, W., AND HOOGERHEIDE, J. C. 22 ROStENBERG, A. 3., AND NISAN, B.- 19491938 Dehydrogenation reactions by suS-urlSaction AND oxydasq depensions of Cl. sporogenes. Biochem. J., Si8ur soaction e-tmino-acide oxydasique de32, 437-448 * *'poro.e=8 et CZ..accharobuty.cum en

10. HoOGERHEIDE, J. C., ANDKocHo*ATY, W. pr6sence d'oxyg~ne. Biochim. et Biophys.1938 Reduction of amino-acids with gas- Acta, 3, 348-357.

23. NismAN, B., AND VINET, G. 1950 Le m6ca-e hgen bysuspensions, *of9Cl. nisme enzymatiquede la reaction de desami-

11. WOODS, Dih. JAND C9FTON9 C. E. 1937 nation couplle chez lea bact6ries ankrobies11.Wo's D. D., ADCITNC. E.....1937........ strictes du groupe de Cl. sporogenes. Ann.Hydrogen production and amino acid utili- ist.rPateur, 78 1 1.zation by Cl. tetanomorphum. Biochem. J. 2 istPsuB. 19 Ld at dg31, 1774-1788. 23a. NIXON, B. 1949 La degradation du glu-

12. CuITON, C. E. 1940 The utilization of cose et de sodium en aerobiose par les sue-amino-acids and of glucose by Cl. botulinum. pensions de Cl. 8porogene8 et Cl. saccharo-J. Bacteriol., 39, 485-497. butyricum. Compt. rend., 229, 6335.

13. COHEN-BAZIREz, G., COHEN, G. N., AND 24. NISmAN, B., AND VINET, G. 1949 L'arsenitePnhVOT, A. R. 1948 Nature et mode de de sodium inhibiteur sp6cifique des amino-formation des acides volatils dans les cul- acides reductases des bact6ries ana6robiestures de quelques bacttries ana6robies pro- strictes. Compt. rend., 229, 675-676.


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40 B. NISMAN [VoL. 18

25. AuBEL, E., AND HOUGET, J. 1945 Consom- sur un ana6robie strict. Experientia, 3,mation d'oxygbne par Cl. acCarobutyricum. 107-108.Rev. can. biol., 4, 48497. 39. WIELAND, H., AND PISTOn, H. J. 1936 Vber

26. AuBEL, E., ROSENBERG, A., AND DE CHEZEL- das dehydrierende Enzymststem, von Aceto-LES, N. 1945 Action de l'oxygane sur la bacter peroxydans. I. ttber den Mechanis-degradation et la synth6se des amino-acides mus der oxydationsvorginge. Ann., 522,par Clostridium sporogene8. Rev. can. 116-137.biol., 4, 502-9. 40. NISMAN, B., AND WIESENDANGER, S. B. 1954

27. STADTmN, E. R., AND BARKER, H. A. 1949 La deshydrogenation des acides-acetoniquesFatty acid synthesis by enzyme preparations par les extraits des bacteries anaerobies.of Cl. kiuyveri. II. The aerobic oxidation Compt. rend., 238, 292-295.of ethanol and butyrate with the formation 41. LEHNINGER, A. L. 1951 Phosphorylationof acetyl phosphate. J. Biol. Chem., 180, coupled to oxidation of dihydrophospho-1095-1115. pyridine nucleotide. J. Biol. Chem., 190,

28. STEPHENSON, PM., AND STICKLAND, L. H. 345-9.1931 Hydrogenase. II. The reduction of 42. STILL, J. L., BuELL, M. V., AND GREEN, D. E.sulphate to sulphide by molecular hydrogen. 1950 Studies on the cyclophorase system.Biochem. J., 25, 215-220. IX. Oxidation of L-alanine. Arch. Bio-

29. STEPHENSON, Ms. 1947 Some aspects of chem., 26, 413-419.hydrogen transfer. Antonie van Leeuwen- 43. NISMAN, B. 1950 La degradation du pyru-hoek, J. Microbiol. Serol., 12, 33-48. vate de sodium par des extraits enzyma-

30. Koiriz, H., AND WILSON, P. W. 1951 The tiques de Cl. sporogenes et Cl. saccharobu-comparative biochemistry of molecular tyricum: une reaction phosphoroclastique.hydrogen. In Bacterial physiology. Edited Compt. rend., 230, 248-250.by Werkman, C. H., and Wilson, P. W. 44. MAGER, J., AND NIsON, B. 1953 Los L-Academic Press, New York, N. Y. glutamo-deshydrogenases de Cl. sporogenes

31. FAuEgS, L., AND FISCHER, E. 1947 On the et Cl. saccharobutyricum. Compt. rend.activation of molecular hydrogen by Proteus (in press).vulgaris. J. Biol. Chem., 167, 787-805. 45. GALE, E. F. 1948 The nitrogen metabolism

32. NIsXw, B., AND MAGER, J. 1952 Diphos- of gram-positive bacteria. Bull. Johnsphopyridine nucleotide and phosphate Hopkins Hosp., 83, 119-175.requirement for oxidation of amino-acids 48. VON EuLER, H., AND ADLER, E. 1939 Zurby cell-free extracts of obligate anaerobes. sterischen spezifizitlit der Glutaminsaure.Nature, 169, 243-244. Enzymologia, 7, 21-25.

33. NisXON, B., MAGER, J., AND TURPIN, A. 47. VON EuLER, H., ADLER, E., G#NTHER, G., AND1953 Diphosphopyridine nucleotide and ELLOTr, L. 1939 Die isocitronensaure-phosphate requirement for oxidation of dehydrase und Glutaminsiuresynthese isamino-acids by cell-free extracts of obligate h6heren Pflanzen und in Hefe. Enzymolo-anaerobes. Biochim. et Biophys. Acta gia, 6, 337-341.(in press). 48. FINCHAM, J. R. S. 1951 The occurrence of

34. MCILWAIN, H. 1948 Preparation of cell-free glutamic dehydrogenase in Neurospora andbacterial extracts with powdered alumina. its apparent absence in certain mutantJ. Gen. Mlcrobiol., 2, 288-291. strains. J. Gen. Microbiol., 5, 793-6.

35. ADLER, E., DAS, N. B., VON EULER, H., AND 49. BRAUNSTEIN, A. E. 1939 The enzyme sys-HEYMANN, U. 1938 Biologische Dehyd- tem of transamination, its mode of actionrierung und Synthese der Glutaminsdure. and biological significance. Nature, 143,Compt. rend. trav. lab., Carlsberg, 22, 15-24. 609-610.

36. ADLER, E., HELLSTR?5M, V., GtNTHER, G., 50. BRAUNSTEIN, A. E., AND AsAnE, R.M. 1945AND VON EULER, H. 1938 t'ber den enzy- The mode of deamination of i-amino-acidsmatischen Abbau und Aufbau der Gluta- in surviving tissues. J. Biol. Chem., 157,minsaure. III. In Bacterium coli. Z. 421-422.physiol. Chem., 255, 14-26. 51. NISMAN, B., COHEN, G. N., RAYNAUD, M.,

37. DEWAN, J. G. 1938 The l(+) glutamic ANDRoSENBERG,A.J. 1947 Etudequanti-dehydrogenase of animal tissues. Biochem. tative du role de l'acide pyruvique et deJ., 32, 1378-1385. l'acide a-cetoglutarique dans l'inhibition

38. AuBEL, E., ROSENBERG, A. J., AND SzUL- de l'amoniog6n~se chez les bacteries anaero-MAsSTER,J. 1947 Action deleau oxygdn~e bies. Bull. soc. chim. biol., 29, 65-655.


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52. SHULL, G. M., AND PETERSON, W. H. 1948 tein der d-AminosAureoxydase. Biochem.The nature of the sporogenes vitamin and Z., 300, 225-239.other factors in the nutrition of Cl. eporo- 68. KENNEDY, E. P., AND BARKER, H. A. 1951genes. Arch. Biochem., 18, 79-88. Butyrate oxidation in the absence of in-

53. GALE, E. F. 1945 Studies on bacterial organic phosphate by Cl. kluyveri. J.amino-acid decarboxylases. 5. The use of Biol. Chem., 191, 419-438.specific decarboxylase preparations in the 69. IJPMANN, F. 1946 Acetyl phosphate. Ad-estimation of amino-acids and in protein vances in Enzymol., 6, 231-267.analysis. Biochem. J., 89, 46-52. 70. REED, L. J., AND DEBUSK, B. G. 1952

54. KAPLAN, N. O., AND CIOTrI, M. M. 1953 Chemical nature of an a-lipoic acid conju-Competitive inhibition of hydroxylamine gate required for oxidation of pyruvate andon alcohol dehydrogenase. J. Biol. Chem., a-ketoglutarate by an E. coli mutant. J.201, 785-794. Biol. Chem., 199, 881-888.

55. KOEPSELL, H. J., AND JOHNSON, M. J. 1942 71. REED, L. J., DEBUsK, B. G., GUNBALUB, I.Dissimilation of pyruvic acid by cell-free C., AND HORNBERGER, C. S., JR. 1951preparations of Cl. butylicum. J. Biol. Crystalline a-lipoic acid: a catalytic agentChem., 145, 379-386. associated with pyruvate dehydrogenase.

56. KOEPSELL, H. J., JOHNSON, M. J., AND MEEK, Science, 114, 93-94.J. S. -1944 R.le of phosphate in pyruvic 72. Koms, S., DEL CAMPILLO, A., GUNSALUS,acid dissimilation by cell-free. extracts of I. C., AND OCHOA, S. 1951 EnzymaticCl. butylicum. J. Biol. Chem., 184, 535-547. synthesis of citric acid. IV. Pyruvate as

57. NISMAN, B., AND MAGER, J. 1952 Coenzyme acetyl-donor. J. Biol. Chem., 198, 721-736.A as a cofactor of the pyruvate dehydro- 72a. CHANTRENNE, H., AND LIPMANN, F. 1950genase system of Cl. saccharobutyricum. Coenzyme A dependence and acetyl donorNature, 169, 709-711. function of the pyruvate-formate exchange

58. STUMPP, P. K. 1945 Pyruvic oxidase of system. J. Biol. Chem., 187, 757-767.Proteus vulgaris. J. Biol. Chem., 169, 73. LYNEN, F., REICERT, E., AND RUEFF, L.529-544. 1951 Zum biologischen Abbau der Essig-

59. MOTED, H. S., AND 0'KANE, D. J. 1952 saure. VI. "Aktivierte Essigsdure," ifrerFractionation of the pyruvate oxidase of isolierung auf Hefe und ihre chemischeProteus vulgaris. J. Biol. Chem., 195, natur. Ann., 574, 1-32.375-381. 74. LYNEN, F., AND REICIE RT, E. 1951 Zur

60. OCHOA, S. 1951 Biological mechanisms of chemischen Struktur der "aktiviertencarboxylation and decarboxylation. Essigslure". Angew. Chem., 68, 47-48.Physiol. Revs., 31, 65-107. 75. LOHMANN, K. 1932 Beitrag zur enzymati-

61. OCHOA, S., STERN, J. R., AND SCHNEIDER, M. schen Unwandlung von synthetischemC. 1951 Enzymatic synthesis of citric Methylglyoxal in Milchshure. Biochem.acid. II. Crystalline condensing enzyme. Z., 254, 332-354.J. Biol. Chem., 198, 691-702. 76. RACKER, E. 1951 The mechanism of action

62. STERN, J. R., SHAPIRO, B., STADTMAN, E. R., of glyoxalase. J. Biol. Chem., 190, 685-696.AND OCHOA, S. 1951 Enzymatic synthesis 77. KORms, S., DEL CAMPILLO, A., AND OCHOA,of citric acid. III. Reversibility and S. 1952 Pyruvate oxidation system ofmechanism. J. Biol. Chem., 198, 703-720. heart muscle. J. Biol. Chem., 195, 541-547.

63. STADTMAN, E. R. 1952 The purification and 78. IJTTLEFELD, J. W., AND SANADI, D. R. 1952properties of phosphotransacetylase. J. R6le of coenzyme A and diphosphopyridineBiol. Chem., 196, 527-534. nucleotide in the oxidation of pyruvate.

64. STADTMAN, E. R. 1952 The net enzymatic J. Biol. Chem., 199, 65-70.synthesis of acetyl-coenzyme A. J. Biol. 79 SCHWEWET, R. S., FULD, M., CHESLOCK, K., ANDChem., 196, 535-546. PAUL, M. H. 1951 Initial stages in pyru-

65. DOIAN, M. I., AND GUNSALUS, I. C. 1951 vate oxidation. In Phosphorus metabolism,Pyruvic acid metabolism. II. An acetoin vol. I, pp. 246-259. The John Hopkinsforming enzyme system in Streptococcus Press, Baltimore, Maryland.faecalis. J. Bacteriol., 62, 199-215. Pres, R. S., aryland.

66. HOLEMAN, A. F. 1904 Notice sur l'action 80. SWEET, R. S., AND CHESLOCK, K. 1952de l'eau oxyg6n6e sur les acides-cetoniques Pyruvic oxidase of pigeon breast muscle.et sur les dicetones. 1. 2. Rec. trav. chim., III. Factors influencing enzymatic activity.23, 169-178. J. Biol. Chem., 199, 749-756.

67. NEGELEIN, E., AND BROMEL, H. 1939 Pro- 81. SEAMAN, G. R. 1953 Role of thioctic acid


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in the transfer of acyl groups. Proc. Soc. culaire. Biochim. et Biophys. Acta, 8,Exptl. Biol. Med., 82, 184-189. 459-465.

82. LIPMANN, F., AND TuTT1z, L. C. 1945 A 86a. NIsMAN, B., COHEN, G. N., WIxSENDANGER,specific micromethod for the determination S. B., AND SzuLmBsTR, J. 1954 La dbg-of acyl-phosphates. J. Biol. Chem., 159, radation du p-hydroxybutyrate par les ex-21-28. traits de Cl. saccharobutyricum. Compt.

83. CHOU, T. C., NovzLL, G. D., STADTMAN, E. rend., 237, 180-1809.R., AND LIPMANN, F. 1950 Fractionation 87. WIESENDANGER, S. B., AND NisSAN, B. 1953of coenzyme A-dependent acetyl transfer Role de l'ATP dans la reaction de deshydro-systems. Federation Proc., 9, 160. genation du pyruvate catalyze par lee

R4. STADTmAN, E. R. 1950 Coenzyme A-de- extraits deCi.saccharobutricum. Biochim.pendent transacetylation and transphos- et Biophys. Acta (in press).phorylation. Federation Proc., 9, 233. 88. LIPMANN, F. 1953 On chemistry and func-

RS. COHEN, G. N., AND COHEN-BAzIRE, G. 1950 tion of coenzyme A. Bacteriol. Revs.,Reduction by molecular hydrogen of aceto- 17, 1-16.acetate to butyrate by butyric acid bac- 89. NIsXON, B. 1953 The acceptor amino-acidteria. Nature, 166, 1077-1078. reducing system. Compt. rend. (in press).

86. COHEN, G. N., AND COREN-BAZIRE, G. 1952 90. MAmirLA. R., MM QUASTEL, J. H. 1953Ptudes our le m~canisme de la fermentation Amino acid interactions in strict anaerobesac6to butylique. IV. R6duction de l'acetyl- (Ci. eporogers). Biochim. et Biophys. Acta,acetate en butyrate par l'hydrogbne mole- 12, 103-120.


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