Allosteric Controls of Amphibolic in Bacteria - …mmbr.asm.org/content/34/1/20.full.pdf · 2006-03-17 · Allosteric Controls of Amphibolic Pathways in Bacteria ... Precursor Activation

BACTERIOLOGICAL REVIEWS, Mar. 1970, p. 20-39Copyright © 1970 American Society for Microbiology

Allosteric Controls of Amphibolic Pathwaysin Bacteria

B. D. SANWALDepartment of Cell Biology, University of Toronto, Toronto 181, Canada

INTRODUCTION............................................ 20

NATURE OF ENZYMATIC CONTROLS..................................... 21

Enzyme Controls in Biosynthetic and Catabolic Pathways.......................... 21

End-Product Control in Amphibolic Pathways................................... 21

Precursor Activation............................................ 23

Energy-Linked Controls ............................................ 24Control of Duplicate Enzymes............................................ 24

Interaction of Feedback Loops in Diverging Channels............................. 25

REDUCED COENZYMES AS CONTROL SIGNALS........................... 27

Regulation of Converging Channels ............................................ 27Regulation of the Citric Acid Cycle by DPNH.................................. 29

Control of Glucogenesis...................................................... 30

Regulation of Oxidative Pentose Pathway.............................. 30

Control of Glycogenesis.............................. 31

Control of Other Enzymes by DPNH ........ ...................... 31NATURE OF ENZYMES IN AMPHIBOLIC PATHWAYS 31

TARGET SUBSTRATES............................................... 33

CRITERIA FOR CONTROL............................................... 34

CONCLUSIONS............................................... 35

LITERATURE CITED............................................... 35

INTRODUCTIONTraditionally, pathways of metabolism have

been classified into two major categories, anabolic(or biosynthetic) and catabolic (or degradative).Davis (21) pointed out, however, that this classifi-cation leaves much to be desired. If one considersthe classical example of a catabolic pathway(namely, glycolysis), the difficulty of nomencla-ture is obvious. Bacteria growing on glucose as a

carbon source utilize glycolytic enzymes as a

catabolic pathway, but when growing on a C3 orC4 organic acid basically the same set of enzymesfunction as an anabolic pathway. To circumventthese ambiguities of classification, Davis (21)coined the term amphibolic to designate pathwaysthat fulfill both an anabolic and a catabolic func-tion. As understood at the present time, amphi-bolic pathways encompass the enzymes of glycoly-sis, the hexose monophosphate pathway, gluco-genesis, and the tricarboxylic acid cycle. Theimportance of amphibolic pathways lies in thefact that they furnish carbon skeletons (e.g.,oxalacetate, phosphoenolpyruvate, etc.) which areutilized not only for biosynthetic purposes butalso for the generation of energy by their completedegradation through the terminal oxidative path-ways. For the efficient operation of such metabolicroutes, bacteria have evolved control mechanismswhich are unique to the enzymes of carbohydrate

metabolism and which ensure a correct, coordi-nated flow of carbon fragments into the biosyn-thetic channels and into the energy-generatingpathways. The purpose of this review is not onlyto enumerate these enzymatic control mechanismsand to point out heuristic generalizations but alsoto examine critically the teleonomic significance ofthe occurrence of diverse types of control loops.

Since this review is primarily directed towardsan examination of the enzymatic controls of bac-terial metabolism, it is useful to point out at thevery outset that controls of carbohydrate metabo-lism found in procaryotes tend to be quite differ-ent and more numerous than those found ineucaryotes. This is probably because bacteria lackthe rigid, compartmentational controls such as are

afforded to the eucaryotic cells by the presence ofmitochondria. Because of these organelles, theglycolytic segment which supplies C3 fragments isphysically separated in eucaryotes from the termi-nal oxidative pathway, and this compartmenta-tion determines to some extent the fate of thecarbon skeleton. Compartmentation is not en-

tirely absent, however, in all bacteria. Manygram-positive and gram-negative forms do havesome semblance of an organized structure (meso-somes) partly comparable to mitochondria (24,95, 96), and the association of some enzymes ofthe tricarboxylic acid cycle with the mesosomes

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CONTROL OF AMPHIBOLIC PATHWAYS

has been reported (78). For orientation regardingthe controls of carbohydrate metabolism ineucaryotes, the reader is referred to an excellentreview by Scrutton and Utter (111). In the follow-ing account, I have made no attempts to sum-marize information which deals with regulation ofamphibolic pathways by genetic repression andderepression mechanisms, except when such in-formation is incidental to and complements thediscussion of allosteric controls.

NATURE OF ENZYMATIC CONTROLSEnzyme Controls in Biosynthetic and

Catabolic PathwaysFor a better appreciation of the complexities of

controls of amphibolic pathways, it is pertinent todiscuss briefly the principles of control of theenzyme activities of strictly biosynthetic andcatabolic pathways. Chronologically, the firstclear-cut evidence for any direct control of theactivity of an enzyme by metabolites came in 1956from the study of the isoleucine-valine biosynthe-tic pathway (127) and the pyrimidine biosyntheticpathway in Escherichia coli (144). In a classicpaper, Umbarger (127) demonstrated that isoleu-cine, the end product of threonine-isoleucinepathway, inhibited the first enzyme (threoninedeaminase, EC 4.2.1.16) of its pathway. This heinterpreted as a negative feedback mechanism. Atthe present time, a large number of enzymes areknown which are susceptible to a negative feed-back inhibition of their activity, and it is a fairgeneralization that in purely biosynthetic path-ways the major control exerted is end product ornegative feedback control (4, 114, 126, 128). Verysophisticated variations of this basic homeostaticmechanism have been evolved by various bacteriaand other organisms. Witness, for example, theoccurrence of cooperative feedback (12, 84), con-certed feedback (20, 88), and cumulative feedback(140)-to name a few-in different biosyntheticpathways.

In direct contrast, however, strictly catabolicsequences do not generally seem to be regulatedby end-product inhibition (40-42, 115). Thestrictly catabolic class of enzymes (21) would bethose which convert diverse, nonessential carbonsources into the intermediates of amphibolicpathways (such as the tricarboxylic acid cycle).Numerous such pathways are known in bacteria(40, 42, 75). Degradation of aromatic rings, (+)camphor, arginine, branched-chain amino acids,purine and pyrimidine bases, and many othercompounds, when used as sole carbon sources, isachieved largely by catabolic routes. In nearly allof these cases, the compound in question is brokendown to an intermediate of the tricarboxylic acid

cycle. A brief discussion of these pathways isgiven by Gibson (34). The activity of initiatingenzymes of the degradative pathways is generallycontrolled by compounds which serve as indica-tors of the energy state of a cell such as inorganicphosphate (Pi), pyrophosphate, and adenine orother purine nucleotides. The biodegradativethreonine deaminase of E. coli (129) and aspartase(EC 4.3.1.1) of Enterobacter aerogenes (136) andBacterium cadaveris (137) are both activated byadenosine-5'-phosphate (AMP). The fermenta-tion of threonine in Clostridium tetanomorphum isalso linked to energy metabolism (82, 125, 135).Histidase (EC 4.3.1.3.), the first enzyme of thehistidine degradative pathway in Pseudomonasaeruginosa (66), is inhibited by pyrophosphate,and this inhibition is relieved by AMP and guano-sine-5'-diphosphate (GDP).The examples given above serve to show that

the biosynthetic and catabolic pathways differmainly in the nature of the regulatory signalutilized for control. In the former, the signal is thespecific end product of the pathway; in the latter,the signal is an ultimate product (or easily inter-convertible products) of energy metabolism com-mon to the diverse catabolic channels.

End-Product Control in Amphibolic PathwaysIt has already been mentioned that amphibolic

pathways serve a catabolic as well as an anabolicfunction. It is understandable, therefore, that thecontrol characteristics specific for the catabolicpathways (feedback by energy indicators) and thebiosynthetic sequences (negative feedback by endproduct) be both represented in amphibolic path-ways. To appreciate this statement fully, theschematic diagram shown in Fig. 1 can be dividedoperationally into four linear segments:

(i) Fructose-6-phosphate -v fructose diphos-phate (FDP) - > phosphoenolpyruvate(PEP)

(ii) PEP -- oxalacetate - aspartate (or malate)(iii) Pyruvate -- -+ -+ acetyl coenzyme A (CoA)(iv) Acetyl-CoA -. citrate -- isocitrate - a-

ketoglutarate (a-KG)

In each of these sequences, the last-named me-tabolite may be considered to be the end productof the pathway starting out from the first-namedintermediate, and, in accord with the principles ofend-product control (127), the last named com-pound causes a negative feedback inhibition of thefirst enzyme of its pathway. All of these enzymesseem to be allosteric, as defined by Monod,Changeux, and Jacob (80), and catalyze reactionswhich are physiologically irreversible. Blangy,Buc, and Monod (8), working with phosphofruc-tokinase of E. coli (EC 2.7.1.11; first enzyme of

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PEP(

t

FIG. 1. Schematic representation of some of theglycolytic, glycogenic, and terminal oxidation segmentsin E. coli and their control. Bold arrows indicate in-hibition and the curved arrows denote activation. Plusand minus signs indicate activation or inhibition,respectively, by the various metabolites.

sequence i), demonstrated that PEP inhibits theactivity of this enzyme in an allosteric manner andthat the inhibition is completely reversed byfructose-6-phosphate. Inhibition of phosphofruc-tokinase by PEP was first demonstrated by Uyedaand Racker (130) in rabbit muscle preparations.In Salmonella typhimurium and E. coli, PEP car-boxylase (EC 4.1.1.31), the first enzyme catalyzingthe formation of oxalacetate, is inhibited by anumber of dicarboxylic acids (18, 70, 85), ofwhich aspartate is the most powerful inhibitor.In bacteria, as also in most other eucaryotes,three enzymes, PEP carboxylase (or, pyruvatecarboxylase in some fungi, bacteria, and mam-mals), malic enzyme, and PEP carboxykinase areall theoretically capable of fixing carbon dioxideto produce oxalacetate. It was demonstrated inS. typhimurium (124) and in E. coli that onlymutants which lack PEP carboxy1 se fail to growon pyruvate or its precursors unless citric acidcycle intermediates are also added to the growth

medium. Thus, it is clear that oxalacetate mustonly be produced in vivo by PEP carboxylase.The third sequence, starting with pyruvate (to

acetyl-CoA), is catalyzed by the well-knownmulti-enzyme complex (38, 59) constituted of pyruvatedehydrogenase (EC 1.2.4.1), dihydrolipoyl trans-acetylase (EC 2.3.1.12), and dihydrolipoyl de-hydrogenase (EC 1.6.4.3.), which act sequentiallyto produce acetyl-CoA. Here again, acetyl-CoAexerts a negative feedback on the first enzyme(pyruvate dehydrogenase) of the complex in E.coli (110).

Finally, it has been shown (142) that a-KG, theend product of the sequence starting with acetyl-CoA, inhibits citrate synthetase (EC 4.1.3.7) ofE. coli and Salmonella (133, 142) in an allostericmanner. While only segments of constitutiveglycolytic channel and citric acid cycle are con-sidered here, it should not be forgotten that newlinks to existing amphibolic sequences are createdwhen bacteria grow on sources of carbon otherthan glucose. As examples, E. coli growing onglycerol dissimilates this compound through aninducible glycerol kinase (EC 2.7.1.30) and a-glycerophosphate dehydrogenase [EC 1.1.1.8 (39,57)]. These enzymes serve as links to the existing(constitutive) triose isomerase (EC 5.3.1.1.) andaldolase (EC 4.1.2.13), and thus a glucogenic seg-ment leading from glycerol to FDP is created,and, as with other examples given before, FDPinhibits by feedback the first enzyme (glycerolkinase) of this pathway (145). Similarly, whenenteric bacteria are growing on a C4 acid as thesole carbon source, pyruvate is produced by de-carboxylation of malate by an inducible triphos-phopyridine nucleotide (TPN)-specific malic en-zyme (EC 1.1.1.40), and this enzyme, in effect,becomes the first enzyme of the pathway leadingfrom malate to acetyl-CoA. In accord with theprinciples of end-product control, acetyl-CoAinhibits the TPN-specific malic enzyme by anallosteric competition with the substrate, malate(108).There is suggestive evidence that end-product

control also operates in the segment between FDPand glucose-6-phosphate during glucogenesis.Bacteria, like higher organisms (119), use the firstenzyme of this sequence, fructose diphosphatase(EC 3.1.3.11) to produce fructose-6-phosphatewhich is then converted into glucose-6-phosphateby hexose isomerase (EC 5.3.1.9). Fructose di-phosphatase is produced constitutively in E. coliand is of no significance whatsoever when glucoseor its precursors are the growth substrates (27),as is clearly demonstrated by the fact that E. colimutants lacking this enzyme grow normally onglucose. Indeed, the functioning of the enzymeduring active glycolysis would be deleterious to

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the organisms because, if left uncontrolled, itwould convert FDP produced by phosphofructo-kinase (Fig. 1) back to fructose-6-phosphate.Part of this problem of metabolic short-circuitingis solved in E. coli, and in higher organisms, byhaving an energy-linked signal (adenine nucleo-tides) produce opposite effects on the two sub-strate-coupled enzymes, phosphofructokinase andfructose diphosphatase. The former enzyme inE. coil is activated (6, 8), and the latter is inhibitedby adenosine diphosphate (ADP) or adenosinemonophosphate [AMP (28)]. However, when thecell is energy saturated (i.e., when the ATP/AMPratio is high), this control system is of minimalsignificance because the ADP and AMP levels areexpected to be low under such conditions (seebelow). Fraenkel (26) recently demonstrated thatin E. coil glucose-6-phosphate inhibits fructosediphosphatase and this negative feedback possiblyrestricts the conversion of FDP to fructose-6-phosphate during glycolysis. This feedback sys-tem is not expected to be effective during growthon a glucogenic substrate (such as glycerol) be-cause the steady-state levels of the inhibitor (glu-cose-6-phosphate) are considerably reduced undersuch conditions (46, 92). This control has beendemonstrated to operate in vivo. Using a mutantof E. coil which lacks both hexose isomerase andglucose-6-phosphate dehydrogenase (EC 1.1.1.49),it was demonstrated (26) that growth of this mu-tant on glycerol is inhibited drastically in thepresence of small amounts of glucose, i.e., underconditions when the concentration of glucose-6-phosphate increases.

Precursor ActivationFrom the physiological point of view, a control

mechanism unique so far to amphibolic pathwaysis precursor activation (43, 106, 109). Opera-tionally, this control is symmetrically opposite ofend-product control. In the case of the latter thelast metabolite of a pathway inhibits the first en-zyme, whereas in precursor control the first me-tabolite of the sequence activates the last enzymeof that sequence. In all known cases, the activatedenzyme catalyzes a reaction which is practicallyirreversible under physiological conditions. Pre-cursor activation was first discovered by Leloirand co-workers (64, 65) who demonstrated thatmammalian glycogen synthetase (EC 2.4.1.11)was powerfully activated by glucose-6-phosphate,an amphibolic precursor of glycogen. Since thena number of such positive "feedforward" loopshave been found in bacteria and some fungi, butonly in amphibolic pathways. In the enteric bac-teria, the intermediate which occupies a centralposition in this regard is FDP. As in the case ofend-product control, one can delineate arbitrarily

a number of metabolic segments where precursoractivation operates.

(i) FDP - PEP - oxalacetate(ii) FDP - PEP pyruvate(iii) PEP pyruvate -+ -4 -+ acetyl-CoA(iv) FDP * fructose-6-phosphate -+ glucose-1-

phosphate -- adenosine diphosphate glu-cose (ADP-glucose)

In each of these cases, the enzyme activated by thefirst-named metabolite is the one preceeding thelast-named intermediate. It is interesting to notethat FDP activates pyruvate kinase (EC 2.7.1.40)(sequence ii) and PEP carboxylase (sequence i),both of which are located at a branch point (Fig.1) and use PEP as a substrate. This circumstance,as will be seen later, creates a number of compli-cations in the regulation of glycolysis for whichthe enteric organisms have evolved ingenious andnovel types of control mechanisms. Because FDPactivates pyruvate kinase from a variety ofsources, including yeast (43) and mammals (7,121, 123), it would seem logical to assume thatthis control may have arisen relatively early dur-ing evolution. Perhaps the same is true of theactivation of ADP-glucose pyrophosphorylase byFDP (sequence iv). Preiss and co-workers (90, 91)demonstrated this control not only in a number ofbacteria (Aerobacter aerogenes, Arthrobacter,Micrococcus lysodeikticus, and E. coli) but also ingreen plants. As was mentioned before, glycogensynthesis in mammalian cells is controlled bymodulation of glycogen synthetase, whereas inbacteria and plants it is controlled by modulationof ADP-glucose pyrophosphorylase which con-verts glucose-1-phosphate into ADP-glucose (36).The latter is the glucosyl donor for a-i ,4-glucansynthesis. The ADP-glucose pyrophosphorylaseof E. coli is activated not only by FDP but also byglyceraldehyde-3-phosphate and PEP, althoughFDP is the activator par excellence. It appearsthat easily interconvertible precursor pools ratherthan a specific precursor are the control signalshere. An entirely analogous situation is known tooccur in some biosynthetic pathways. The regula-tory signal for aspartate transcarbamylase (EC2.1.3.2), the first enzyme of the pyrimidine path-way in E. coli, for instance is provided by thenucleoside triphosphate pools, although cytidine-5'-triphosphate (CTP) is the most potent inhibitor(32, 33).The precursor activation loops discussed above,

although typical of the enteric forms, are not theonly ones found in bacteria. As an illustration ofthis point, lactate dehydrogenase (EC 1.1.1.27)from several members of the genus Streptococcusis strongly and specifically activated (139) byFDP, but this precursor activation does not occur

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in E. coil or Butyribacterium rettgeri (122, 138).This example, among others to be discussed later,illustrates that the "unity of biochemistry" con-cept which has been so useful in the study of otherbiochemical phenomena has paid little dividendsin the field of controls. Unity of principles under-lying controls certainly exists, but the manifesta-tions are necessarily as divergent as the diversityof the organisms.

Energy-Linked ControlsAs stated earlier, energy-linked controls (to the

exclusion of end-product or precursor control)are characteristic of strictly catabolic or degrada-tive pathways. It is, therefore, not surprising thatamphibolic routes which fulfill both a catabolicand anabolic function are also controlled bycompounds which are energy donors or energyacceptors of a cell. Generally speaking, the com-pounds of greatest interest in this regard areAMP, ADP, pyrophosphate, adenosine triphos-phate (ATP), and inorganic phosphate. However,since various other pyrimidine or purine nucleo-side mono-, di-, and triphosphates are readilyinterchangeable with adenine nucleotides bymeans of the ubiquitous nucleoside kinases, mostof which have an equilibrium constant near unity,there is a possibility that nucleotides other thanthe adenylates may serve as indicators of theenergy state of the cell.The amphibolic channels provide not only

hydrogen for the electron transport chain, whichgenerates energy in the form of ATP, but also thecarbon skeletons needed for biosynthesis. TheATP produced is used up by the biosyntheticpathways and other energy-utilizing processes ofthe cell with the generation of ADP or AMP.Thus, when the supply of energy is low adjust-ments must occur to speed up the ATP-generatingreactions and, conversely, when the energy supplyis high these reactions must be slowed down.Bacteria, like other organisms (3, 61), achievethis by modulating the activities of crucial, rate-limiting steps of the sequences which are involvedin ATP generation (see Fig. 1). Thus, in E. coli,phosphofructokinase is activated by ADP andGDP (8), one of the two pyruvate kinases (seelater) is activated by AMP (72), pyruvate de-hydrogenase component of pyruvate dehydro-genase complex is activated by GDP and AMP(110), and fructose diphosphatase is inhibited byAMP and ADP (28). The significance of activa-tion by AMP (or ADP) of the first three enzymesmentioned above lies in the fact that for the regu-lation of glycolytic and terminal oxidation se-quence, AMP (and to a lesser extent, ADP), asfirst suggested by Krebs (61), is a sensitive indi-cator of the state of ATP supply because of the

intervention of adenylate kinase (EC 2.7.4.3).This enzyme is present in large concentrations inmost microbial cells and has an equilibrium con-stant [(AMP) (ATP)/(ADP)2j of 0.44 (23). If thetotal concentration of adenylates in a bacterialcell, such as that of E. coli, is assumed to be 4 mM,the concentration of ATP, ADP, and AMP equili-brium will be 3, 0.9, and 0.1 mm, respectively.It can be readily calculated that when the concen-tration ofATP drops to 2 mm (33%), the concen-tration ofADP rises to 1.5 mm (67%) and that ofAMP rises to 0.5 mm (500%). It is reasonable toassume, then, that the level of AMP, more thanthe absolute concentration ofATP or ATP/ADP,is a sensitive control signal (61). Any decrease inthe supply of ATP will be reflected in the stimula-tion of enzymes which are involved either directlyin the generation of ATP (pyruvate kinase) orwhich furnish products which may be used forenergy generation through the terminal oxidativepathway. Atkinson (5) recently suggested that abalance between the concentrations of the adenyl-ates may be an important regulatory parame-ter for all enzymes which consume or produceATP whether or not they have regulatory sitesaccommodating specific adenine nucleotides.

Control of Duplicate EnzymesSo far, the discussion has centered around the

generalities of signals utilized for the control ofenzymes of the amphibolic pathways. At thispoint, it is necessary to digress a little and examinethe strategy employed by the cells to manage theproblems of metabolic traffic which arise when thecells switch over from growth conditions underwhich glycolysis predominates (growth on glu-cose) to those under which glucogenesis is essen-tial (for instance, growth on C3 or C4 com-pounds). Glycolysis shares with glucogenesis theblock of freely reversible, constitutive enzymesproducing FDP from PEP. For glucogenesis tooccur, the bacterial cells induce enzyme systemswhich lead to the formation of PEP. In the entericbacteria, the two enzymes which are capable ofdoing so are PEP carboxykinase (EC 4.1.1.32) andPEP synthase; the former is induced (45, 143)when a C4 compound (succinate, malate) servesas a carbon source and the latter only when a C3compound (alanine, pyruvate, or lactate) is theenergy source (17). In both cases, PEP is producedat the expense of ATP. Since the cells containpyruvate kinase, PEP produced during glucogene-sis could easily be converted to pyruvate and ATPinstead of giving rise to triose phosphate. Thiswould be wasteful not only because the synthesisof hexoses would be curtailed but also becausethere would be an unnecessary accumulation ofpyruvate which during glucogenesis is produced

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by the TPN-specific malic enzyme. This enzyme isknown to be derepressed during growth of E. colion succinate or malate (48; 103). To prevent thiskind of short-circuiting, the enteric bacteria, likemammalian tissues (120), produce two pyruvatekinases, one activated by FDP and derepressedduring growth on glucose (74) and the otheractivated by AMP and produced constitutively(Waygood and Sanwal, unpublished data). TheFDP-activated pyruvate kinase probably does notfunction during growth on succinate (i.e., duringglucogenesis) because of repression and alsobecause the concentration of the activator (FDP),like other hexose phosphates, falls to a level al-most 25% of that found during vigorous growthon glucose (46; Sanwal, unpublished data). Simi-larly, the AMP-activated pyruvate kinase func-tions during glucogenesis under restrictive condi-tions because AMP activation in molecular termsmay be considered equivalent to ATP inhibition,and the functioning of this pyruvate kinase is thusdependent on the energy balance of the cell.A second instance in which duplication of en-

zymes occurs in the enteric bacteria is in the con-version of malate to pyruvate. The two enzymesinvolved are the TPN- and the diphosphopyridine(DPN)-specific (EC 1.1.1.38) malic enzymes (48).The equilibrium constants of both of the enzymesare such that they favor formation of pyruvate,and this is also their probable function in vivo.As with the two pyruvate kinases discussed above,again the presence of two malic enzymes may beviewed as a device which directs an orderly meta-bolic flow depending upon the carbon sourcebeing utilized by the cells. Thus, when glycolysis ispredominant, the primary source of pyruvate(and ATP, see below) is PEP, and it is desirable toprevent an uncontrolled flow of the same com-pound (pyruvate) into the central pool throughmalate. This is accomplished in E. coli by therepression of the TPN-specific malic enzyme (103)during growth on glucose (catabolite repression)and the inhibition of its activity by reduced di-phosphopyridine nucleotide (DPNH) (104), acompound which accumulates during glycolysis(Table 1) and, as we will see presently, is a centralcontrol signal for a larger number of metabolicsequences. For the same possible reasons, theactivity of the second, constitutive, malic enzyme(DPN-dependent) is inhibited by CoA and thisinhibition is relieved specifically by aspartate (98).In physiological terms, this means that the DPN-specific malic enzyme is formed by the entericbacteria for the degradation of C4 acids and itfunctions only when there is an accumulation ofthese compounds. The amount of C4 acids de-graded through this enzyme, however, is furtherregulated by the state of the energy supply of the

TABLE 1. Concentration ofDPN and DPNH underdifferent conditions ofgrowths

Growth phase Carbon source DPN DPNH DPN/DPNH

Early log Glucose 2.5 5.5 0.45Succinate 3.2 3.2 1.00

Mid log Glucose 2.5 3.0 0.82Succinate 2.9 2.0 1.45

Stationary Glucose 2.1 4.5 0.48Succinate 3.2 3.1 1.00

a E. coli $ cells were preadapted to glucose orsuccinate by growing them for at least 10 genera-tions in the appropriate medium. Fernbach flaskscontaining 1 liter of mineral salts medium (withglucose or succinate as carbon source) were inocu-lated with a suspension of pregrown cells andshaken vigorously at 37 C. Growth was monitoredby measuring cell density in a Klett photometer.At appropriate times, 500 ml of the culture wascentrifuged and analyzed for coenzyme content byenzymatic procedures. Values in the table arereported as micromoles per gram, dry weight.

cell, which is reflected in the inhibition of theenzyme by ATP in a manner that cannot berelieved by the activator aspartate (98). It is notclear at this stage, however, why the presence oftwo enzymes is required to achieve the same end,unless it be that one of the enzymes (TPN depend-ent) has a function in addition to the formation ofpyruvate, namely, the generation of reducedtriphosphorpyridine nucleotide (TPNH) for bio-synthetic purposes.Recent evidence indicates that duplicity of

enzymes also occurs in the initial segment of thetricarboxylic acid cycle. Two TPN-specific iso-citrate dehydrogenases (EC 1.1.1.42) have beenreported to occur in Salmonella and E. coli, one ofwhich is inhibited by ATP, ADP, or guanosinetriphosphate (GTP) (77). Two TPN-dependentglutamate dehydrogenases (EC 1.4.1.3.) have alsobeen separated from E. coli (Lo and Sanwal,unpublished data). As was pointed out by Um-barger and Brown (128, 129), and as has been theexperience so far, whenever two enzymes arefound to catalyze the same reaction in a cell theirmetabolic roles are quite different, and it is likelythat the duplicate enzymes referred to above willbe found to be regulated differently from eachother.

Interaction of Feedback Loops in DivergingChannels

It was mentioned at the outset that the inter-mediates of amphibolic pathways serve a numberof functions and some of these are not entirelyrelated. This will be clear from a study of Fig. 2.An intermediary metabolite such as PEP for

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TCA Cycle - >6falutsmi

Arinirm-Asp gin.

Lyuins

Purims

FIG. 2. Cascade type of regulation of glycolysis in

E. coil. Plus and minus signs indicate activation or

inhibition, respectively, by the various metabolites.

instance, not only is used for the biosynthesis of

the aromatic amino acids (tryptophan, tyrosine,

phenylalanine) but also must be converted into

pyruvate, on the one hand, through the catabolic

channel (catalyzed by pyruvate kinase), and to

oxalacetate, on the other, through the anapleroticchannel (catalyzed by PEP carboxylase). In other

words, PEP must be used to satisfy both the

energy and biosynthetic requirements of the cells.

Now, biosynthesis can only proceed at a constant

rate if there is sufficient supply of energy (in the

form of ATP) and reducing power. Since the

supply of ATP must decrease as energy-consum-

ing, biosynthetic processes proceed and since the

energy supply can only be replenished by the

oxidation of C3 compounds, it follows that the

amounts of PEP withdrawn via the catabolic

channel must fluctuate considerably. This de-

mands that mechanisms should be available

which control the distribution of PEP into the

anaplerotic and the catabolic channels. In the

enteric bacteria, the enzymes which produceoxalacetate and pyruvate are both controlled in

an allosteric manner by the substrate (PEP),which typically yields sigmnoid saturation curves

(72, 73). The Km, of PEP is not the same, however,for the two enzymes. For PEP carboxylase, it is

10 mm (101) and for the FDP-activated pyru-vate kinase it is _ 3.5 mm (Waygood and Sanwal,unpublished data). This means that PEP carboxyl-ase must be virtually inactive in the cell. Followingthe work of Keech and Utter (52, 53) with ATP-dependent pyruvate carboxylase (EC 6.4.1.1.) (ofanimals), it has now been demonstrated that inthe enteric bacteria (10, 70), acetyl-CoA activatesPEP-carboxylase powerfully; at saturation itbrings the Km for PEP down to 1 mm. Activationof PEP carboxylase seems to be a mechanismwhich ensures that a supply of oxalacetate will beforthcoming to enable the oxidation of acetyl-CoA (by condensation with oxalacetate to citrate)through the citric acid cycle. However, oxalace-tate is required not only for condensation withacetyl-CoA but, provided that energy is available,also for the biosynthesis of proteins, pyrimidines,and purines after its conversion to aspartate byaspartate-glutamate transaminase (EC 2.6.1.1).Now, in a bacterial cell, the levels of acetyl-CoAmust be highly variable because not only is thiscompound diverted to the synthesis of fats but itsown synthesis by the pyruvate dehydrogenasecomplex is modulated (see Fig. 2) by the state ofenergy supply. (AMP activates pyruvate dehydro-genase component and in molecular terms this isequivalent to inhibition by ATP.) If activationonly by acetyl-CoA were an indispensable condi-tion for the production of oxalacetate, a constancyof its supply commensurate to the demands ofprotein, pyrimidine, and purine biosynthesiscould not be maintained. However, activation byFDP of PEP carboxylase, discussed earlier, per-haps alleviates this difficulty. It can be shown thatacetyl-CoA and FDP together bring about acooperative activation (73) of PEP carboxylase;i.e., the percentage activation in the presence ofacetyl-CoA and FDP together is much higherthan the sum of percentages of activation in thepresence of each activator alone (Fig. 3). Thismeans, in physiological terms, that FDP com-pensates for any decrease in the degree of activa-tion of PEP carboxylase brought about by adecrease in the supply of acetyl-CoA, and viceversa. In other words, the cooperative activationis a necessary stabilizing device as far as the for-mation of oxalacetate is concerned, and helps toensure that the biosynthetic function of glycolysisis not seriously impeded when momentary adjust-ments are occurring in the competing catabolicchannels in response to changes in energy levels.To clarify this a little further, consider a situationwhere momentarily the ATP supply is high andacetyl-CoA is being converted into fatty acids.Under these conditions, the right-hand channelsof Fig. 2 leading from FDP via PEP to acetyl-CoA are probably not functioning optimally be-

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mM FDP

FIG. 3. Interactions offructose-i, 6-di-P and acetyl-CoA in the activation of PEP carboxylase from Sal-monella typhimurium. The numbers above the linesrepresent the concentration ofacetyl-CoA (73).

cause they require ADP (phosphofructokinase) orAMP (pyruvate dehydrogenase system and AMP-activated pyruvate kinase) for maximal activity.As was pointed out earlier, when ATP concentra-tion is high the concentration of ADP and AMPis low. As the concentration of ATP decreases inproportion to acetyl-CoA consumed in fatty acidsynthesis, the concentration of AMP and ADPrises and triggers a cascade-type of activation ofthe catabolic channels due to the combined actionof strategic, interdependent, energy-linked, andprecursor activation loops (Fig. 2). Thus, ADPactivates phosphofructokinase, which results inenhanced levels of FDP. It is expected that thisaugmented concentration ofFDP will compensatefor the momentary decreases in the concentrationof acetyl-CoA in the stimulation of PEP carboxyl-ase. At the same time, the excess FDP activatesone of the two pyruvate kinases (FDP-activated)and AMP activates the other one. The PEPformed from FDP activates the pyruvate dehydro-genase complex along with AMP with the netresult that both ATP (from pyruvate kinase) andacetyl-CoA levels are replenished. Again, thedecrease in the FDP concentration is compensatedfor by augmented levels of acetyl-CoA in theactivation of PEP carboxylase. It is recognized inthis discussion that what has been stated above is,in our present state of knowledge, only a reason-able guess and has necessitated some ad hoc as-

sumptions. For instance, the concentration ofATP in vivo may momentarily decrease not onlybecause of the utilization of acetyl-CoA but alsobecause of the operation of other ATP consuming

biosynthetic processes, and one can easily visua-lize a situation in which the concentrations ofFDP and acetyl-CoA decrease simultaneously.Further safeguards seem to have been providedfor such eventualities to ensure functioning of thebiosynthetic arm. Thus, PEP carboxylase is acti-vated by CMP, cytidine diphosphate (CDP), andGTP (but not by ATP, ADP, or AMP), and thisactivation, like FDP, is again cooperative withacetyl-CoA (101). It is not entirely clear at thepresent time what relationship the cytidine nucleo-tides may have to the metabolic fluctuations in thelevels of acetyl-CoA which necessitate the occur-rence of the aforementioned cooperative activa-tion. This fact, however, does not deter from theimportance of the argument that when a metabo-lite is used for both biosynthetic and catabolicpurposes, and when, of necessity, the amount ofthat metabolite passing through the catabolicchannel fluctuates in response to energy adjust-ments, controls, however complex, have to bemade available which ensure that a constantamount of the shared metabolite goes into thebiosynthetic channel. This argument has heuristicvalue. Bacteria do differ with regard to the loca-tion of branch points in amphibolic pathways. InPseudomonas and Arthrobacter, PEP carboxylaseseems to be absent and oxalacetate is produced, asin yeast (68) and animals (52), by the ATP-dependent pyruvate carboxylase. It is at thesepoints that novel types of controls are likely to befound.

It was stated earlier that all of the activatingcontrols of PEP carboxylase of enteric bacteriahave their origin in the fact that this enzymewould otherwise be virtually nonfunctional invivo because of its low affinity for PEP. One mightask why evolution has selected for an enzyme ofthis nature. A reasonable answer is perhaps to befound in the relative ease with which the presenceof such an enzyme allows bacteria to switch fromone carbon source to another. PEP carboxylase isonly important when growth occurs on glucose,glycerol, or pyruvate, but it is a hindrance when aC4 compound is being utilized. Because of thealready high Km for PEP, it is relatively easy toinhibit the enzyme almost completely by a nega-tive feedback of its activity by C4-dicarboxylicacids (73; Fig. 2).

REDUCED COENZYMES AS CONTROLSIGNALS

Regulation of Converging ChannelsIt must be amply clear from the above account

that quite an unorthodox strategy is employed bythe cells to control metabolic flow in divergingchannels. Simple metabolic control mechanisms

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such as end-product inhibition, precursor activa-tion, etc. are not sufficiently sophisticated to fulfillregulatory demands at branch points ofamphibolic pathways. As will be seen presently,the same seems to hold true for converging se-quences. In the enteric bacteria, a convergingsequence consists of enzyme systems which leadfrom oxalacetate to pyruvate via malate dehydro-genase (EC 1.1.1.37) and malic enzyme (Fig. 1,4). The problem of control in this area can best beunderstood, if the question is asked: when oxal-acetate is produced by PEP carboxylase in re-sponse to a particular need, namely, to enableoxidation of acetyl-CoA or to supply the bio-synthetic routes with aspartate, what mechanismdetermines that it will only be utilized for thepurpose it was produced? In the absence of such amechanism, oxalacetate could conceivably beeasily diverted to malate and thence to pyruvate(through the converging channel; Fig. 4), and thelatter would then be available for the generationof more acetyl-CoA. This would be wasteful inview of the fact that a controlled quantity ofpyruvate is already produced from PEP by pyru-vate kinase. The problem as outlined here israther unique to bacteria because, here, compart-mentation appears to be in a relatively rudimen-tary form. In eucaryotes, the same difficulty inregulation exists but it seems to have been over-come by imposing rigid compartmentation ofcertain enzymes and metabolites inside mitochon-dria. Consider the formation of oxalacetate inmitochondria when acetyl-CoA levels are high. Ashas been mentioned before, activation of the ATP-dependent pyruvate carboxylase by acetyl-CoAresults in the formation of enough oxalacetate topermit the oxidation of acetyl-CoA (52). The re-sulting excess oxalacetate is probably not utilizedby mitochondrial malate dehydrogenase owing toits inhibition by the substrate, nor is it immedi-ately accessible to the extramitochondrial malatedehydrogenase owing to the problems associatedwith permeation, etc. (14, 113). This sort of com-partmentation reasonably assures that the surplusof oxalacetate will mostly be utilized for the pur-pose it was produced, namely, to condense withacetyl-CoA. Admittedly, this is an extremely sim-plified version of the actual, and definitely moreelaborate, controls available in the partitioningof metabolites (113) between mitochondria andcytosol, but it serves to illustrate the fact that inthe absence of compartmentation the problemsof enzymatic regulation in this area are unique tobacteria.

Recently it has been found that the main con-trol signal in converging channels in E. coli andS. typhimurium is DPNH. This coenzyme specifi-cally inhibits, in an allosteric manner, the activity

FIG. 4. Enzymic reactions inhibited by DPNH(shown by heavy arrow):1 = pyruvate kinase; 2 =PEP carboxylase; 3 = PEP carboxykinase; 4 =malate dehydrogenase; 5 = TPN-speciflc malic en-zyme; 6 = pyruvate dehydrogenase complex; 7 =citrate synthetase; 8 = PEP synihase.

of malate dehydrogenase and malic enzyme alongwith that of a large number of other enzymes (seebelow). If oxalacetate produced in response toactivation of PEP carboxylase by acetyl-CoA andFDP is to be prevented from passing into theconverging channel (Fig. 2, 4), malate dehydro-genase and malic enzyme must be blocked. Themalate dehydrogenase of E. coli seems to bedesigned in such a way that, in the presence ofincreasing concentrations of DPNH, a continuallysteep sigmoidal velocity response is given byoxalacetate as a substrate with a concomitantdecrease in the Vmaz of the reaction (97). Thismeans, in physiological terms, that provided theDPNH concentration is high, oxalacetate has athreshold concentration below which it cannot beeasily converted to malate. Even more stringent isthe control of malic enzyme (103) which is allo-sterically inhibited by oxalacetate (Ki apparent =0.01 mM), DPNH, TPNH, acetyl-CoA and cyclic3'-5'-AMP (105). The importance of the inhibi-tion of this enzyme by oxalacetate is obvious inview of what has been discussed before about thelack of rigid compartmentation and its possibleconsequences. Thus, when oxalacetate concentra-tion is high in response to a specific metabolicdemand, inhibition of competing enzymes prob-

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ably ensures the use of oxalacetate to fulfill thatdemand.

Regulation of the Citric Acid Cycle by DPNHIt was suggested by Atkinson (3) that the entry

of acetyl-CoA into the citric acid cycle may beregulated by ATP. The suggestion was based onthe finding that mammalian (47) and yeast citratesynthetases (87) are inhibited by this compound.This has been found to be true also for somestrictly aerobic bacteria like Bacillus subtilis (25).In the enteric forms, however, the citric acid cycleseems to be controlled by DPNH. Weitzman (131)first showed that citrate synthetase of E. coli ispowerfully inhibited by the reduced coenzyme in aspecific manner. With the finding that malatedehydrogenase is also inhibited by DPNH (97),there is very little doubt that DPNH indeed maybe the central control signal for the regulation ofcitric acid cycle. It may then be asked why DPNHis, to the exclusion of other possible compounds(such as adenine nucleotides which are the energyindicators of a cell), an allosteric inhibitor of thetwo crucial enzymes of the energy-generatingpathway. It has been suggested (131, 132) thatthis inhibition may be a kind of feedback controlby end product, since DPNH is generally theproduct of the dehydrogenating reactions of thecitric acid cycle. This explanation is probably notthe right one for the enteric bacteria because,first, so far as is known, DPN is reduced only atone step of the cycle, namely, the step catalyzed bya-ketoglutarate dehydrogenase (EC 1.2.4.2) and,second, as will be discussed later, DPNH inhibitsin an allosteric manner the activity of severalother enzymes which have no connection whatso-ever with the citric acid cycle (Fig. 4).The reason for the inhibition of citric acid cycle

by DPNH has rather to be sought in a biochemi-cal oddity of the enteric bacteria and probablyother facultative anaerobes as well. Despite theextensive use of E. coil in the study of intermedi-ary metabolism and biochemical phenomena ingeneral, this organism is atypical so far as itsterminal respiration is concerned. The entericbacteria live largely by anaerobic glycolysis evenwhen growing on glucose aerobically. The citricacid cycle predominantly serves a biosyntheticfunction under these circumstances and is perhapsinterrupted at the level of succinic dehydrogenase(EC 1.3.9.1). These statements are based on theobservation that a number of crucial enzymessuch as succinic dehydrogenase, succinic thio-kinase (EC 6.2.1.5), a-ketoglutarate dehydro-genase, and cytochromes are repressed in thepresence of glucose (2, 93). Mutants which lacksuccinic dehydrogenase (44) ubiquinones andvitamin K [required for oxidative phosphoryla-

tion (19)] grow well aerobically on glucose. It isfor these reasons, also, that unlike eucaryotes andpossibly other strictly aerobic bacteria, the entericorganisms have a preponderance of DPNH whenaerobic growth occurs on glucose. It can be dem-onstrated that the DPN/DPNH ratio is on theaverage 0.5 in glucose-grown cells as comparedwith 1.0 in the case of succinate-grown cells(Table 1). The level of DPNH can thus be con-sidered as an indicator of the state of glycolysis.Since ATP must obviously be generated, albeit inan uneconomical manner, primarily by glycolysisduring growth on glucose, it is easy to see thepurpose of the inhibition of citrate synthetase byDPNH. This inhibition would minimize the pro-duction of more DPNH, through a-ketoglutaratedehydrogenase, for example, under conditions inwhich a supply of this compound is already avail-able in excess of that which can be handled by therepressed electron transport chain. However, itmay be noted that functioning of citrate synthe-tase is important, if for nothing else to provideglutamate by further transformations of citrate,which in turn must be available to aminate oxal-acetate. Accordingly, it has been found thatacetyl-CoA and oxalacetate deinhibit the enzymeagainst DPNH and a-ketoglutarate, respectively(142).Before going any further, a discussion as to why

E. coli and related facultative anaerobes haveevolved an apparently wasteful way of utilizingglucose aerobically, namely, by glycolysis alone,would be useful. Amarsingham and Davis (2)advanced the ingenious argument that this modeof growth gives selective advantage to the entericbacteria in competition with other microbes.Thus, incompletely oxidized intermediates towhich the competing organisms may not adaptreadily are quickly produced from glucose, andthese may remain in the external milieu for use assoon as the supply of glucose is exhausted.Most of the arguments concerning regulation so

far given have, of necessity, been based on obser-vations made with the enteric bacteria, if only forthe reasons that these organisms have been popu-lar with biochemists and physiologists alike andthat a level of information is available whichallows some reasonable guesses to be made re-garding integration of control circuits. Neverthe-less, the question may be asked whether controlby DPNH is a general property of bacterial sys-tems. This question also relates to the contentionmade earlier that part of the complex controlsutilizing DPNH as inhibitor are necessitated inbacteria by the absence of rigid compartmenta-tional controls. In a recent study, Weitzman andJones (134), using inhibition of citrate synthetaseby DPNH as a criterion, demonstrated that all of

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the 18 genera of gram-negative bacteria (includingAzotobacter, Pseudomonas, Moraxella, Flavobac-terium, etc.) tested had a citrate synthetase sus-ceptible to inhibition, whereas the enzyme fromaerobic gram-positive bacteria (among whichare Micrococcus, Staphylococcus, Bacillus, andArthrobacter) was not affected by DPNH. Withinthe DPNH-susceptible class, subclasses could bedifferentiated on the basis of the capability ofAMP to reverse DPNH inhibition. It has beensuggested (134) that the enzyme systems of bac-teria showing relatively simple mesosomal or-ganization may be more susceptible to DPNHinhibition, whereas those associated with complexmesosomal organization are not affected byDPNH. There is little information available,however, about details of mesosomal structure inbacteria, such as is needed to document such aproposal.

Control of GlucogenesisGlucogenesis from C3 and C4 compounds in

both eucaryotes and procaryotes is made possibleby the presence of enzyme systems other thanpyruvate kinase which convert a number of com-pounds to PEP (Fig. 4). It is now well establishedthat the ubiquitous pyruvate kinase is incapable ofproducing PEP from pyruvate owing to its nearirreversibility. In E. coli the two main enzymeswhich make glucogenesis possible from C4 sub-strates are PEP carboxykinase and TPN-specificmalic enzyme, both of which are induced to aconsiderable extent during growth on C4 com-pounds such as succinate and malate (103, 143).Loss of PEP carboxykinase by mutation preventsgrowth on succinate, malate, fumarate, and ace-tate, which proves the indispensibility of thisenzyme for glucogenesis on these substrates (45).Such mutants, however, grow on pyruvate orlactate and it has been now demonstrated byCooper and Kornberg (17) that this is madepossible by the induction ofPEP synthase which isthe main glucogenic route during growth on C3compounds.

It was shown recently that both PEP carboxy-kinase (143) and TPN-specific malic enzyme (103,104) are inhibited in an allosteric manner byDPNH. Whereas the latter enzyme is also suscep-tible to inhibition by TPNH, acetyl-CoA, andcyclic 3', 5'-AMP (103, 105), the inhibition of theformer enzyme by DPNH is strictly specific (143).The specificity of the control for the two enzymes,of course, reflects the specificity of their functions.Whereas the function of PEP carboxykinase isrestricted exclusively to the formation of PEP byan ATP-dependent decarboxylation of oxalace-tate, the functions of malic enzyme are diverse, asfor instance, formation of TPNH for reductive

synthesis and formation of acetyl-CoA via pyru-vate during glucogenesis. The allosteric control ofthe glucogenic enzymes by DPNH can be con-sidered as a mechanism whereby unnecessaryglucogenesis is prevented when vigorous growthis occurring on glucose. It has been pointed outbefore that in the enteric bacteria levels ofDPNHare indicators of the state of glycolysis, and itstands to reason that they be also the signalswhich hinder the functioning of enzymatic routeswhich are opposite to glycolysis.

Regulation of Oxidative Pentose PathwayOne of the enigmatic problems perhaps central

to the understanding of carbohydrate metabolismin bacteria is the nature of the control system(or systems) which determines the distribution ofglucose-6-phosphate into the pentose phosphatepathway, on the one hand, and the glycolyticpathway on the other. It is well known that pen-tose is synthesized in bacteria by both the oxida-tive and the nonoxidative pathways. In theformer, 1 mole of glucose-6-phosphate is con-verted to 1 mole of pentose, 1 mole of carbondioxide, and 2 moles of TPNH, whereas in thelatter 5 moles of hexose phosphate is convertednonoxidatively into 6 moles of pentose phosphate.Numerous studies with "4C-labeled glucose (50,51) have demonstrated that 20 to 30% of glucoseis utilized by the enteric bacteria through theoxidative pentose pathway. There is a distinctpossibility, however, that the major function ofthis pathway is not to generate pentose for bio-synthetic purposes. This statement is based on thefinding, among others, of Caprioli and Rittenberg(11) that nucleosides isolated from cells of E. coligrown on 1-_80-glucose have all of the 180 labelin the 5'-position. Similarly, Katz and Rognstad(49) showed that only a very small amount ofglucose carbon passing through the oxidativepathway in E. coil is converted into phosphori-bosyl pyrophosphate. In view of these observa-tions, there is a distinct possibility that one of theprimary functions of the oxidative pentose path-way is to generate TPNH for reductive biosynthe-sis. Model and Rittenberg (79), on the basis oftheir finding that the amount of glucose metabo-lized through the oxidative pathway drops toabout 50% during stationary compared to thelogarithmic phase, have suggested that the oxida-tive pathway is regulated by the availability ofoxidized TPN, and this is also the opinion of otherworkers (22). It has now been demonstrated (99)that the activity of glucose-6-phosphate dehydro-genase of E. coli is inhibited in an allostericmanner by DPNH. It is indeed extremely interest-ing that in E. coli, DPNH controls all of theTPNH-generating pathways of the cell. The two

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main enzymes that produce TPNH, namely,TPN-specific malic enzyme and glucose-6-phos-phate dehydrogenase, are directly inhibited byDPNH, and the activity of the third enzyme,TPN-specific isocitrate dehydrogenase (E. coil notpossessing a DPN-specific enzyme), is indirectlycontrolled by an inhibition of citrate synthetasewhich is the only enzyme supplying isocitrate inthe enteric bacteria. It is germane to point out,however, that the control of glucose-6-phosphatedehydrogenase by DPNH is not of universaloccurrence in bacteria. In P. aeruginosa, for in-stance, this enzyme shows completely differentcharacteristics and is inhibited by ATP, much likethe enzyme from mammalian tissues.

Control of GlycogenesisGlycogenesis, as mentioned elsewhere, is con-

trolled in bacteria at the level of ADP-glucosesynthesis (36). In animals, it is controlled both atthe level of glycogen synthesis and breakdown(glycogenolysis) by the regulation of glycogen syn-thetase (65) and phosphorylase (EC 2.4.1.1),respectively. The glycogen phosphorylase in bac-teria, to the contrary, seems not to be regulated byallosteric mechanisms at all (16). The importance,again, of reduced pyridine nucleotide coenzymesin the control of glycogenesis in E. coil is demon-strated by the recent finding of Preiss and co-workers (91) that ADP-glucose pyrophosphoryl-ase is powerfully activated by TPNH. This controlis exerted on the enzyme in addition to that causedby triose phosphates discussed earlier. Preiss andco-workers (91) view the activation by TPNH as adevice by which the flux of carbon is divertedtowards glycogen synthesis during limited growth,such as is expected to occur in the stationaryphase during which the concentration ofTPNH isexpected to increase because it is not consumed inbiosynthetic reactions.

Control of Other Enzymes by DPNHIt is clear from the account given above that the

reduced pyridine nucleotide coenzymes play alarge part in the regulation of amphibolic path-ways. Indeed, the enzymes discussed before arenot the only ones controlled by reduced coen-zymes. Phosphotransacetylase (EC 2.3.1.8) fromE. colt, for instance, is powerfully inhibited byDPNH (116) and, judging from the kinetic evi-dence presented by Suzuki et al. (116), this seemsto be an allosteric type of inhibition. Similarly, ithas been shown that (37) the dihydrolipoyf de-hydrogenase component of the pyruvate dehydro-genase complex of E. coli is inhibited by DPNH.In this case, however, it is not clear whether this isproduct inhibition (a property of all enzymes be-

cause the substrate and the product are expectedto bind at the same site on the enzyme surface andcause inhibition by steric hinderance) or allostericinhibition.Much of the information about reduced pyri-

dine coenzyme-linked controls has been accumu-lated primarily from a study of enzymes of E. coliand other enteric bacteria. It does, however, seemthat unconventional controls of this nature will befound to be of more common occurrence thanhitherto realized. As an example, it has recentlybeen demonstrated that the reductive pentosecycle in Hydrogenomonas (69) and in Rhodopseu-domonas spheroides and R. rubrum (94) iscontrolled by DPNH through the activation ofribulose-5-phosphate kinase (EC 2.7.1.47). Fur-ther work must be done with these systems beforethe importance of the DPNH effect can be eval-uated.

NATURE OF ENZYMES IN AMPHIBOLICPATHWAYS

Having discussed the physiological aspects ofcontrol mechanisms so far, the important problemremaining is the nature of enzymes which serve asreceptors of control signals. Two properties maybe considered to characterize the regulatory en-zymes sufficiently: (i) the presence of a distinctand relatively specific site on the enzyme whichbinds the regulatory ligand and (ii) the sigmoidal(or cooperative) velocity response given by most,if not all, of these enzymes to increasing concen-tration of substrate under certain conditions. Inregard to the latter property, the allosteric en-zymes of the amphibolic pathways can be divided(Fig. 5) into two clear-cut categories (Table 2):those which show modulator-independent co-operativity (MIC systems), i.e., which yield

MIC MDC

FiG. 5. Typical plots of rate-concentration data ob-tained with MJC (modulator-independent cooperative)and MDC (modulator-dependent cooperative) sys-tems in the absence (indicated by 0) and presence(indicated by I) of inhibitors.

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TABLE 2. Nature of allosteric enzymes in amphibolic pathways of E. coli

Enzyme Classification Inhibitor Activators Target substrate References

ADPG-pyrophospho- MDC (?) AMP FDP, PEP, 90, 91rylase triose-P

Citrate synthetase MDC DPNH, a-KG K+ (?) OAA (DPNH), 131, 142acetyl-CoA,(a-KG)"

Glucose-6-phosphate MDC DPNH Spermidine TPN 99dehydrogenase

DPN-specific malate MDC DPNH Oxalacetate, 97dehydrogenase malate

DPN-specific malic MDC CoA, ATP Aspartate Malate 98enzyme

TPN-specific malic MDC DPNH, TPNH NH3 Malate 103-105enzyme oxalacetate,

acetyl-CoA,3'-5'-AMP

PEP carboxylase MIC Aspartate, Acetyl-CoA, PEP 10, 18, 100-102malate FDP,

GTP,CDP

PEP carboxykinase MDC DPNH Oxalacetate 143Phosphofructokinase MIC PEP ADP, GDP Fructose-6- 6, 8

phosphatePhosphotransacetylase MDC DPNH, ATP Pyruvate Acetyl phos- 116

phatePyruvate dehydrogenase MIC (?) Acetyl-CoA AMP, GDP, Pyruvate 110

PEPPyruvate kinase (FDP MIC FDP PEP 72, 74

activated)

See explanation in the text.

sigmoidal rate-concentration plots in the absenceof inhibitors and activators, and those which yieldsigmoidal initial velocity plots only in theirpresence [modulator dependent cooperativity(MDC)]. It is not necessary to confine this classi-fication to the enzymes of the amphibolic path-ways. With a few exceptions, all allosteric en-zymes known so far in diverse pathways can beplaced in one or the other of these categories. Tothis extent also, the discussion which follows isindeed applicable to all allosteric enzymes.Although the physiological advantages accruing

to an organism by possessing enzymes which givesigmoidal rate-concentration curves are obvious,the actual molecular mechanism underlying thisbehavior is certainly not known. Starting with theearly proposals of Monod et al. (81) for the MICsystems, the trend is to analyze the kinetic be-havior of allosteric enzymes in terms of subunitinteractions, whether they involve simple isomeri-zation or association-dissociation of the subunits(4, 29, 114). According to Monod's hypothesis,cooperativity is caused by the differential bindingof substrates to two conformational states of theprotein which are in equilibrium determined by adimensionless constant, L. If this constant is such

that most of the protein exists in a state which hasvery low affinity for the substrate (state T, ortight), cooperativity ensues because the substratepulls the enzyme towards a state (R, or relaxed)which has much higher affinity for it. In thistheory, for reasons of symmetry, hybrid statesbetween T and R are considered almost nonexist-ent. Other hypotheses (4, 56), however, do notassume equilibrium between T and R states butconsider that conformation of individual subunitscan be changed by binding of ligand such thatforms are possible where one subunit is in the Rstate and another in the T state.As applied to the kinetic data, however, there is

no reason a priori to consider sigmoidal velocityresponse as resulting exclusively from subunitinteractions. A number of authors (107, 114, 141)have interpreted cooperativity of substrate withsome enzymes ofMIC category on the basis of thepresence of two kinds of substrate binding sites onthe enzyme surface, or even on the basis of thepresence of alternate modes of binding of sub-strate molecules to single active sites (117). Muchof this difference of opinion can be traced to thenature of techniques used in arriving at conclu-sions. Subunit isomerizations can only be detected

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by physicochemical methods, such as equilib-rium dialysis or stopped-flow techniques. Be-cause varying degrees of nonhyperbolic characterof rate-concentration plots can arise by changesin magnitudes of rate constants associated withvarious reactants, kinetic studies are least suitedfor deducing the nature of allosteric mechanismswith any degree of certainty. As an illustration,phosphofructokinase of E. coli (8) and muscle(76), both of which are MIC systems, behavekinetically in a way which is explicable on thebasis of the Monod model. However, for themuscle enzyme at least, cooperativity of the sub-strate is not discernible in equilibrium ligand-binding studies (54), which perhaps means thatpart of the cooperativity observed in initial veloc-ity studies, at the minimum, must arise fromkinetic interactions, i.e., through modifications ofcertain critical rate constants (71, 114, 141).Similar uncertainties exist in the case of anotherglycolytic enzyme, glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12) from yeast. Analysisof temperature-jump kinetics indicates (55) thatfor this enzyme cooperativity can be explained onthe basis of Monod's model. However, using theformation of enzyme-substrate complex and rateof product release as criteria, another group ofworkers (15) consider cooperativity to be due tothe presence of unproductive binding sites for thesubstrate on the enzyme surface.To some extent, the same kinds of uncertainties

of interpretation existing with MIC systems alsoapply to the MDC systems (71, 104, 143), but theinterpretation is comparatively simpler with thelatter category of enzymes. It may be recalled thatthe rate-concentration plots in MDC systems arealways hyperbolic in the absence of inhibitors, butonly become sigmoidal in their presence. Thisbehavior necessarily means that the catalytic sitesmust all be equal and function independently ofeach other in the absence of the inhibitors. Verysimply, the sigmoidality of the rate-concentrationplots may then be explained on the basis of theisomerization of subunits (104) in the presence ofthe inhibitor. One can assume, for instance, thatout of two final states of the subunits (R and T)possible, an enzyme constituted of identical sub-units exists primarily in the R state (a state whichhas high affinity for substrate). The inhibitorbinds at a specific regulatory site and converts thesubunits to the T state which has low affinity forthe substrate. With these assumptions, it can bereadily realized (i) that without the inhibitor(since state T is almost nonexistent) the substratewill give hyperbolic rate-concentration plots be-cause only the high affinity state is available; (ii)that in the presence of unsaturating concentra-tions of inhibitor (since the enzyme is now distrib-

uted into R and T states) the substrate will give asigmoid response because it now binds with differ-ent affinities to the two states; (iii) that when theinhibitor is saturating and all of the enzyme is inthe T state the substrate will again give a hyper-bolic response, because it is now able to bind(albeit, with low affinity) only to the available Tstate; and (iv) that the inhibitor despite its struc-tural dissimilarity will give competitive inhibitionagainst substrate, because the former stabilizes theT state and the latter the R state and a push-pullsituation will develop.The actual experimental data obtained with

MDC systems are compatible with this modelonly for the case of malic enzyme (104) andcitrate synthetase (142), the inhibitors of whichgive competitive inhibition against at least one ofthe substrates. For malate dehydrogenase andPEP carboxykinase, however, the inhibition (97,143) is noncompetitive. This fact raises the possi-bility that either cooperativity of the substratearises in these cases due to kinetic interactions(i.e., changes of rate constants by the binding ofthe inhibitor) or the velocity constants for productrelease of R and T states are quite different.

TARGET SUBSTRATESIt is clear from what has been said above that in

the present state of our knowledge only reason-able guesses can be made about the mode of ac-tion of allosteric enzymes. Indeed, in the abovediscussion the regulatory enzymes have beentreated as if they had only one substrate, a situa-tion which occurs only rarely. Most of the regula-tory enzymes catalyze reactions in which two ormore substrates participate, and this introduces alarge number of complications in kinetic analyses;the greater the number of substrates and modula-tors, the greater becomes the possibility that anyone single mechanism may not be able to explainthe kinetic intricacies. Whatever the kineticmechanism, however, the finding of greater physi-ological interest is that for most of the MIC andMDC systems of enzymes, it is generally onlyone of the two or more substrates which gives asigmoidal velocity response. As an example, phos-phofructokinase (MIC system) has two sub-strates, ATP and fructose-6-phosphate, and onlythe latter yields cooperative rate-concentrationplots. Similarly, for malic enzyme (MDC system),only one of the substrates, malate, gives a sig-moidal velocity response in the presence ofdiverseinhibitors. Such a substrate may be termed aphysiological target substrate because it is verylikely that the.=currence of an allosteric controlon the enzyme is directed towards the distributionof that particular substrate through the modu-lated pathway. Sigmoidality has the inherent ad-

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vantage that the affinity (the reciprocal ofMichaelis constant) of the substrate for the en-zyme can be decreased (or increased) sharply overa narrow threshold range, and this characteristicmakes possible a sensitive control system.At first glance, the concept of a target substrate

may seem trivial because a majority of bireactantallosteric enzymes utilize ATP (or ADP) or one ofthe coenzymes, DPN or TPN, as the second sub-strate. Since these nucleotides are shared by alarge number of enzymes, common sense suggeststhat no useful purpose would be served by havingthese compounds as target substrates. It is physio-logically meaningful always to have the non-nucleotide substrate as the target substrate, andthis is also the actual experimental finding (Table2). There are, however, a number of regulatoryenzymes, both in amphibolic as well as other bio-synthetic pathways, which do not utilize the nu-cleotides as the second substrate and in which thefinding of a target substrate has considerablephysiological significance. As an example, onemay consider the case of citrate synthetase of E.coli (142; Wright and Sanwal, unpublished data)which has two substrates, acetyl-CoA and oxal-acetate, and two allosteric inhibitors, DPNH anda-KG. Since citrate synthetase belongs to theMDC system, both substrates yield Michaelis-Menten kinetics in the absence of the inhibitors.With a-KG as inhibitor, the initial velocity plotswith oxalacetate as a variable substrate remainhyperbolic (142) but acetyl-CoA plots becomesigmoidal, whereas acetyl-CoA plots remainhyperbolic but oxalacetate plots become sig-moidal in the presence of DPNH. Physiologicallyspeaking, acetyl-CoA is the target substrate fora-KG as oxalacetate is for DPNH; i.e., the inhi-bition by DPNH in the latter case perhaps pri-marily affects the distribution of oxalacetatethrough the modulated enzyme. Whereas both ofthe inhibitors will block formation of citrate, theconcentration of oxalacetate will only rise steeply(however momentarily) in the presence of DPNHand that of acetyl-CoA will do so only in thepresence of a-KG. Indeed, this sophisticated con-trol device may be used in vivo to control effi-ciently the fate of certain compounds such asacetyl-CoA in the enteric bacteria. It is wellknown that in animals tricarboxylic acids deter-mine the fate of acetyl-CoA by activating acetyl-CoA carboxylase (EC 6.4.1.2.), the first enzymeof fatty acid biosynthesis (9, 89). In E. coli how-ever, no activators or inhibitors of this enzymehave been found (1), and it seems quite possiblethat here the distribution of acetyl-CoA is solelydetermined by feedback inhibition of citrate syn-thetase by a-KG. This inhibition would not onlycut off the synthesis of a-KG but would also

afford a steeply increasing supply of acetyl-CoAto the fatty acid synthesizing system.

CRITERIA FOR CONTROLThe implicit assumption that has been made in

the discussion of controls of amphibolic pathwaysin bacteria is that the in vitro findings apply toconditions in vivo. This may not always be so, andto this extent the whole discussion may be con-sidered conjectural until all the postulated con-trols have been demonstrated in situ in bacterialcells. Biochemical technology, however, has notprogressed far enough to attempt this approach.Nevertheless, certain criteria must be available bywhich to judge whether inhibition or activation ofan isolated enzyme in vitro plays the same roleinside the cells. One such reasonable criterion isto show that the enzyme possesses an allostericsite for the modulator. This can be achieved byactual desensitization (80) of the enzyme to theeffect of the modulator by physical or chemicaltreatments. A number of enzymes, however, arerefractory to desensitization. If such enzymesbelong to the MDC category, presence of an allo-steric site can be clearly inferred from the kineticdata, because there is no conceivable way togenerate sigmoidal rate-concentration plots of thesubstrate in the absence of an allosteric ligand-binding site.There are, however, no compelling reasons to

consider that only enzymes which carry regulatorysites are important in the control of intermediarymetabolism. Interactions of two or more metabo-lites at the catalytic site of enzymes may contrib-ute significantly to overall regulation of path-ways. Atkinson (5) recently proposed that to asignificant degree amphibolic pathways may beregulated by the energy charge (defined as ATP +0.5 ADP/ATP + ADP + AMP) of the adenylatepools by interactions at the catalytic sites of en-zymes and, furthermore, these interactions may bemodulated in the case of regulatory enzymes bythe binding of allosteric effectors. Although a con-siderable amount of indirect evidence supportsthis hypothesis, it would clearly be impossible atthe present time to demonstrate these events inthe cells.

Despite the difficulties of demonstrating con-trols in vivo, approaches are still available in bac-terial systems which could profitably be used forthis purpose. In E. coli for instance, mutants areavailable (58) which have a thermolabile adenyl-ate kinase. It should be possible in such strainsto measure steady-state concentrations of metab-olites which are suspected of being regulated byenergy-linked controls (such as FDP, acetyl-CoA, pyruvate, etc.) under conditions in whichadenylate kinase is fully active and inactive. Sim-

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ilarly, cold-sensitive mutants are available (86)and others could possibly be made which showaltered control of specific regulatory circuits. Infortunate circumstances, it may even be possibleto select for regulatory mutants not showing anyconditional phenotype. Cattaneo et al. (13) andGovons et al. (35) recently selected mutants of E.coli which accumulate glycogen (judged by theintensity of iodine staining), and this has beenshown to result from altered regulatory properties,of ADP-glucose pyrophosphorylase which in themutant shows a much lower affinity for theinhibitor (AMP) and much higher affinity for theactivator (FDP) compared to the enzyme fromwild-type E. colt. Yet another approach withenteric bacteria, particularly E. coli, consists inthe treatment of the cells with chelating agentssuch as ethylenediaminetetraacetic acid (62, 63).In this way, cells can be made permeable to anumber of phosphorylated compounds (ATP,ADP, etc.) and control properties of enzymes canbe measured in situ. This approach has been usedby Liersch and Preiss (67) to assay ADP-glucosepyrophosphorylase and to study its control char-acteristics in vivo.

CONCLUSIONSThe discussions of controls in amphibolic

pathways in bacteria have so far been a mixture ofa number of facts and a large amount of specula-tion. Although the speculations themselves willhave to be subjected in the future to the rigorousscrutiny of experimentation and may or may nothold ground, the factual information itself raisesthe question whether any heuristic generalizationsare at all possible from the study of the controls ofone organism or a group of organisms which maybe applicable to other groups of organisms. Theanswer seems to be that the "unity of biochemis-try" concept which was so useful for the study ofintermediary metabolism per se in diverse organ-isms may not hold so far as coordination andcontrol are concerned. A case in point, for in-stance, is the regulation of citrate synthetase andthe consequent possibilities of partition of acetyl-CoA into the citric acid cycle or the pathway offatty acid biosynthesis. In animals (47), yeast (87),and B. subtilis (25), this enzyme is inhibited byATP. In E. coli, on the other hand, citrate syn-thetase is inhibited by DPNH and a-KG. Still, inanother group of aerobic gram-positive bacteria,the enzyme is not regulated by ATP or DPNH(134), so that these organisms will possibly befound to have quite different control mechanismsfor restricting the flow of acetyl-CoA into differ-ent channels. It seems that control mechanismshave evolved by selection to suit the needs of aparticular organism growing in its habitual milieu.

Such being the case, diversity rather than a nar-row unity would be the desirable feature ofcontrol.One may conclude this essay by pointing out

the problems that remain to be solved in the areaof control of amphibolic pathways in bacteria.One obvious problem, albeit not too exciting, isthe comparative biochemistry of the feedbacksystems in groups or tribes of bacteria. These mayyield valuable clues to taxonomic relationshipsamong different forms (134). Since most of thecontemporary knowledge about controls is basedon E. col, one of the attempts of this review hasbeen to predict possible control points in otherbacteria from an analysis of existing knowledge.Study of these enzymes may unearth systemswhich have properties amenable to detailedphysicochemical analysis. It is the study of thisfundamental area that alone will lead to advancesin our understanding of allosteric phenomena. Todate, essentially only three enzymes, aspartatetranscarbamylase from E. colt (32), glutaminesynthetase (EC 6.3.1.2) from E. coil (140), andbovine glutamate dehydrogenase (30), are knownwhich have the prerequisites, namely, ease of iso-lation in large quantities, susceptibility to de-sensitization, and possibilities for enzymatic orchemical modifications of amino acid residues,for further work to be profitable.

ACKNOWLEDGMENTS

Investigations in the author's laboratory were supportediniually by the National Research Council of Canada at theUniversity of Manitoba, and later by the Medical Research Coun-cil at the University of Toronto. I deeply appreciate the help ofTheodore Lo, E. B. Waygood, and H. Duckworth in the prepa-ration of the manuscript.

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