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Trehalose has also been isolated from lichens and

algae, and is present in many higher plants, such as

the resurrection plant (Selaginella lepidophylla), and

Arabidopsis thaliana. It is also a component of the

wound exudates of some plants, such as Fraxsinus

aras. Many different species of bacteria, including

Streptomyces, Mycobacterium, Corynebacteria,

Rhizobium, Arthrobacter,andEscherichia have been

shown to contain trehalose, and in most cases to

also have the ability to synthesize this disaccharide.

These many studies suggest that trehalose is probably

present in many, if not most bacteria.

In the animal kingdom, trehalose was ﬁrst reported in

insects where it is present in hemolymph and also in

larvae or pupae. In the adult insect, the levels of

trehalose fall rapidly during certain energy-requiring

activities such as ﬂight, suggesting a role for this sugar as

a source of glucose for producing ATP. In addition to

insects, trehalose has also been identiﬁed in the eggs of

the roundworm, Ascaris lumbricoides, where it may be

present at levels as high as 8% of the dry weight. It is

also found in a number of other invertebrates. On the

other hand, trehalose has not been found in any

mammals, although the enzyme trehalase is present in

human intestine (intestinal villae membranes) and in

human kidney (kidney brush border membranes). The

intestinal enzyme is probably important in metabolizing

ingested trehalose, since this sugar is present in various

foods such as mushrooms which contain signiﬁcant

amounts of trehalose. On the other hand, the role of

trehalase in human kidney remains a mystery.

Many lower organisms do contain the enzyme

trehalase, and this activity may be important in

maintaining physiological concentrations of trehalose

in the cytoplasm. That is, as discussed below, trehalose

plays a role as a protectant or stabilizer in some cells,

and the levels of trehalose in these cells is signiﬁcantly

increased during times of stress. A high concentration of

trehalose in the cytoplasm could upset the osmolar

balance in the cell and cause serious problems. Thus,

trehalase may be necessary to control such imbalances.

This enzyme may also have an important function in the

utilization of trehalose as an energy and carbon source

for insect ﬂight, for spore germination, and for other

glucose-requiring processes.

In yeast, there are several different trehalases which

apparently have different functions. The cytoplasmic

trehalase, also referred to as the neutral trehalase

because it has a pH optimum of 7.0, is a regulatory

enzyme. This enzyme can exist in an inactive or

zymogenic form, and this zymogen is converted by a

cyclic-AMP-dependent phosphorylation to the active

trehalase. Another trehalase in these cells is the acidic

enzyme, which is found in the vacuoles and has a pH

optimum of , 4.5. The acid enzyme may be involved in

the utilization of trehalose as a carbon source by yeast

since deletion of the gene for this protein leads to an

inability of yeast to grow on trehalose. On the other

hand, the trehalase that is regulated by phosphorylation

might be involved in regulating the levels of trehalose in

the cytoplasm, since trehalose levels can be signiﬁcantly

increased by various types of stress. However, the exact

role of the various trehalase activities still remains to be

determined.

Physiological Functions

of Trehalose

Trehalose may have a number of different functions in

nature, and its speciﬁc role may vary depending upon the

system or organism being considered. The established

functions of trehalose are outlined below.

ASASOURCE OF ENERGY

AND/OR

CARBON

Trehalose levels may vary greatly in certain cells

depending on the stage of growth, the nutritional state

of the organism, and the environmental conditions

prevailing at the time of measurement. Trehalose is the

major sugar in the hemolymph and thorax muscle of

insects, and it is converted to glucose and consumed

during ﬂight. It is also an important source of energy and

carbon in fungal spores and is utilized during the

germination process.

ASASTABILIZER AND PROTECTANT OF

PROTEINS AND MEMBRANES AGAINST

VARIOUS STRESSES

Various organisms, including plants, yeast, fungal

spores, nematodes, brine shrimp, etc., can withstand

dehydration and remain in this state (anhydrobiosis) for

extended periods of time until water becomes available.

These organisms generally contain high concentrations

of trehalose. For example, when nematodes are slowly

dehydrated, they convert as much as 20% of their

dry weight into trehalose. Similar results have been

FIGURE 1 Structure of the naturally occurring isomer of trehalose,

i.e.,

1,1-D-glucopyranosyl-D-glucopyranoside.

252 TREHALOSE METABOLISM

demonstrated with other organisms. The use of treha-

lose to enable cells to survive dehydration may be an

ancient adaptation since even Archaebacteria have been

found to accumulate trehalose in response to stress.

Two primary changes are thought to cause destabi-

lization of lipid bilayers during dehydration. These

changes are fusion and lipid-phase transitions. Trehalose

inhibits fusion and depresses the phase transition

temperature of the dry lipids which maintains them in

the liquid crystalline phase in the absence of water. This

stabilizing effect of trehalose is a property of its structure

and its stereochemistry. X-ray diffraction studies show

that trehalose ﬁts well between the polar head groups of

the lipids with multiple sites of interaction. Its stereo-

chemistry allows it to have the most favorable ﬁt with

the polar head groups of the phospholipids of cellular

membranes.

This ability to withstand stress by increasing the levels

of trehalose has been used by researchers to bioengineer

more stable plant and animal cells. For example,

introducing the genes that code for the biosynthetic

trehalose enzymes (i.e., TPS and TPP) into human

ﬁbroblasts allowed these cells to be maintained in the

dry state for up to 5 days, whereas control cells were

O-PP-uridine

OPO

2–

OPO

2–

TPP

TPS

Malto-oligosaccharides

TreZ

TreY

FIGURE 2 Pathways of biosynthesis of trehalose. The best-known pathway involves the transfer of glucose from UDP-glucose to glucose-6-

phosphate to form trehalose-6-phosphate and UDP, catalyzed by trehalose-phosphate synthase (TPS). A second enzyme in this pathway, trehalose-

phosphate phosphatase (TPP), removes the phosphate to give free trehalose. An alternate pathway involves the enzyme, trehalose synthase (TS)

which catalyzes the interconversion of maltose and trehalose. Another alternate pathway involves two enzymes, TreY and TreZ, which catalyze the

formation of trehalose at the reducing end of a malto-oligosaccharide (TreY) and then hydrolysis of this trehalose (TreZ) to give free trehalose.

TREHALOSE METABOLISM 253

rapidly killed by this treatment. Similar technology has

also been used to produce more stable transgenic plants.

ASAPROTECTANT AGAINST HEAT

In yeast, stimuli that trigger the heat-shock response also

cause an accumulation of trehalose. Two of the subunits

of the trehalose-6-P synthase complex are actively

synthesized when yeast cells are subjected to heat

shock. Yeast mutants that are defective in either of the

genes coding for TPS or TPP are not able to accumulate

trehalose, and as a result, they are much more sensitive

to heat shock than wild-type cells. Physiological

concentrations of trehalose, up to 0.5 M, were found

to protect enzymes of yeast and other organisms from

heat inactivation, in vitro, and trehalose was a consider-

ably better stabilizer than a number of other polyols,

sugars, or amino acids. These data strongly implicate

trehalose as playing a key role in thermotolerance, as

well as in other stress conditions.

ASAFREE RADICAL SCAVENGER TO

PREVENT OXIDATIVE STRESS

Another role for trehalose is in protecting cells against

oxygen radicals. Exposure of yeast to a mild heat shock,

or to a proteosome inhibitor, induced the accumulation

of trehalose but also markedly increased the viability of

these cells during exposure to a free radical-generating

system (such as H

/iron). However, when the cells

were returned to the normal growth temperature, both

the trehalose content and the resistance to oxygen stress

decreased rapidly and returned to the normal level.

Mutants that were defective in the enzymes of trehalose

metabolism were much more susceptible to oxygen

stress than the wild-type organism, but adding exogen-

ous trehalose to the medium enhanced the resistance of

these mutant cells to the oxygen stress. The major effect

of oxygen radicals on these cells was to damage amino

acids in cellular proteins, and trehalose was able to

prevent this damage, suggesting that this disaccharide

functions as a free-radical scavanger.

ASASTRUCTURAL COMPONENT

OF THE

BACTERIAL CELL WALL

In mycobacteria and corynebacteria (and perhaps other

organisms), trehalose is the basic component of a

number of cell wall glycolipids. The best known and

most completely studied of these trehalose lipids is

cord factor, a cell wall component of the tubercle

bacillus, that contains the unusual fatty acid called

mycolic acid, esteriﬁed to the hydroxymethyl group of

each glucose to give trehalose-dimycolate. This lipid is

considered to be one of the major toxic components of

the Mycobacterium tuberculosis cell wall, and is also

largely responsible for the low permeability of this cell

wall, which provides the organism with considerable

resistance to many drugs. However, the function of this

lipid, besides its obvious structural role, is still uncertain.

There are other antigenic glycolipids in the myco-

bacterial cell wall that also have trehalose as the basic

structure. For example, there are a variety of acylated-

trehalose compounds that contain any of three major

types of fatty acids attached to the 2 and 3 hydroxyl

groups of the two glucose moieties. These fatty acids

are unusual and may be either C

16 – 19

saturated fatty

acids, C

21 – 25

-methyl branched fatty acids, or C

24 – 28

-methyl branched,

-hydroxy fatty acids. M. tubercu-

losis and other mycobacteria also have trehalose lipids

that contain sulfate, such as 2,3,6,6

-tetra-acyl-2-sulfate-

trehalose (sulfatide 1), or other types of fatty acids such

as phthienoic acids. This great variation in the types of

fatty acids found in these organisms and as cell wall

components suggests speciﬁc functions, but thus far

these have not been demonstrated.

Finally, some mycobacteria, such as M. kansasii, are

characterized by the presence of seven species-speciﬁc

neutral lipooligosaccharide antigens. These oligoscchar-

ide structures all have a common tetraglucose core

which is distinguished by an

-trehalose substituent to

which are attached various sugars, such as xylose, 3-0-

methylrhamnose, fucose and a novel N-acylsugar. The

exact structures of these complex polymers have not

been established, but speciﬁc lipooligosaccharides are

present in a number of “atypical” mycobacteria.

FURTHER READING

Banaroudj, N., Lee, D. H., and Goldberg, A. L. (2001). Trehalose

accumulation during cellular stress protects cells and cellular

proteins from damage by oxygen radicals. J. Biol. Chem. 276,

24261–24267.

Birch, G. G. (1963). Trehaloses. Adv. Carbohyd. Chem. 18, 201–224.

Brennan, P. J., and Nikaido, H. (1995). The envelope of mycobacteria.

Annu. Rev. Biochem. 64, 29–63.

Crowe, J., Crowe, L., and Chapman, D. (1984). Preservation of

membranes in anhydrobiotic organisms. The role of trehalose.

Science 223, 209–217.

Elbein, A. D. (1967). Carbohydrate metabolism in Streptomycetes.

Isolation and enzymatic synthesis of trehalose. J. Bacteriol. 94,

1520–1524.

Elbein, A. D., Pan, Y. T., Pastuszak, I., and Carroll, J. D. (2003). New

insights on trehalose: A multifunctional molecule. Glycobiology

13, 17R–27R.

Lederer, E. (1976). Cord factor and related trehalose esters. Chem.

Phys. Lipids 16, 91–106.

Leopold, A. C. (1986). Membranes, Metabolism and Dry Organisms,

pp. 377. Cornell University Press, Ithaca, NY.

Pan, Y. T., Carroll, J. D., and Elbein, A. D. (2002). Trehalose-

phosphate synthase of Mycobacterium tuberculosis. Eur.

J. Biochem. 269, 6091– 6100.

Thevelein, J. M. (1984). Regulation of trehalose metabolism in fungi.

Microbiol. Rev. 48, 42–59.

BIOGRAPHY

Alan Elbein is Professor and Chairman of the Department

of Biochemistry and Molecular Biology at the University of

Arkansas for Medical Sciences in Little Rock. He received his

Ph.D. in microbial biochemistry from Purdue University and did

postdoctoral research at the University of Michigan and the

University of California at Berkeley. He joined the faculty as

Assistant Professor of biology at Rice University and then moved to

the Department of Biochemistry at the University of Texas

Health Science Center at San Antonio as Professor. His research

focuses on the role of complex carbohydrates in glycoprotein

function, and on novel carbohydrate target sites in chemotherapy of

microbiol diseases.

TREHALOSE METABOLISM 255

Tricarboxylic Acid Cycle

Richard L. Veech

National Institutes of Health, Rockville, Maryland, USA

Thetricarboxylicacidcycleisthecentralpathwayin

intermediary metabolism, which accounts for the ﬁnal

combustion of foodstuffs into CO

and water while conserving

the reducing equivalents produced for transmission up the

mitochondrial electron transport system, to form the mito-

chondrial proton gradient responsible for ATP generation. The

cycle occurs in nearly all life forms, even the archebacteria,

suggesting that during the early evolution of life its function

was anaplerotic and its direction reversed from that which

exists in an oxygen-containing atmosphere.

The Discoverer of the Cycle

The tricarboxylic acid (TCA) cycle was ﬁrst correctly

formulated by Han Krebs, who was born on August 25,

1900 in Hildesheim. He was the son of a surgeon, Georg,

and mother Alma, who was to die of depression

when Krebs was 19. After completing gymnasium in

Hildesheim in September 1918, Krebs joined the German

army signal corps until the armistice of November 11,

1918. After completing his training in internal medicine

at Gottingen, Freiburg, Munich, and Berlin in December

1925, Krebs looked for ways to obtain biochemical

training. The time spent in the clinics had convinced him

that it was necessary to pursue medical research.

Through the inﬂuence of Bruno Mendel, who served

with Krebs in theThird Medical Clinic in Berlin, and who

was an acquaintance of Albert Einstein, Professor of

Physics at the University of Berlin, who was in turn a

frequent dinner guest at the home of Otto Warburg’s

father, Mendel heard that Warburg was looking for an

assistant and recommended Krebs. This circle of

inﬂuence allowed Krebs to obtain his ﬁrst paid position

as an assistant to Otto Warburg at the Kaiser Wilhelm

Institut fu

r Biologie at Berlin-Dahlem (see Figure 1). This

stint was to change Krebs from a physician to a

biochemist. Of Warburg, Krebs wrote (1981):

Otto Warburg was the most remarkable person I have

ever been closely associated with. Remarkable as a

scientiﬁc genius of the highest caliber, as a highly

independent, penetrating thinker, as an eccentric who

shaped his life with determination and without fear,

according to his own ideas and ideals

The Kaiser Wilhelm Institute was the leading

scientiﬁc research facility of its time. In addition to

Warburg, its faculty included Emil Fischer, Richard

Willstater, Max von Laue, Fritz Haber, Otto Hahn, Lisa

Meitner, Michael Polanyi. Otto Meyerhof, a former

student of Warburg, was working in the same building

and elucidated the glycolytic pathway responsible for

the breakdown of glucose to lactate. Students of

biochemistry who passed through Kaiser Wilhelm

included, among others, Fritz Lipmann and Severo

Ochoa, both of whom were to play a critical role in

showing the role of coenzyme A in the TCA cycle. In

addition to the discipline and rigor required for scientiﬁc

research, Krebs learned from Warburg the techniques he

had developed: use of tissue slices, spectrophotometry

and manometry. These techniques, most notably mano-

metry, were fundamentally important in leading Krebs

to formulate the concept of a metabolic cycle, ﬁrst the

urea cycle and later the TCA cycle. It is important to

recognize in understanding Krebs’ work that the

Warburg dictum “methods are everything” was domi-

nant. There was no theoretical master plan. Only during

his later work on the thermodynamics linking of cellular

redox and phosphorylation states would Krebs remark

in relation to that work, “This is the ﬁrst time I ever

thought you could predict in biology.” In his early work,

Krebs was a thorough empiricist. He could measure the

rates of reaction. He added various potential partici-

pants in a metabolic pathway, and if they increased the

overall rate, then he concluded they were part of the

pathway. Krebs’ training is of central importance to

understand the cycle. As his biographer Holmes has

pointed out, “Hans Krebs took 31 years to become a

well-trained independent scientiﬁc investigator, and only

9 months to make one of the most signiﬁcant discoveries

of his generation in his chosen ﬁeld.”

History of the Discovery

of the TCA Cycle

After leaving Warburg’s lab in 1931, Krebs had obtained

a position as chief medical resident in the university

hospital at Freiburg, and was in charge of caring for 44

patients. With his Warburg manometer and the enzyme

urease, he could measure the production of urea in liver

slices. In his “off time” working by himself with the

assistance of a medical student, Kurt Henseleit within 1

year he had formulated a salts solution to mimic the

composition of plasma, “Krebs–Henseleit saline,” the

basis for modern tissue culture ﬂuids, and had worked

out the ornithine cycle responsible for the hepatic

synthesis of urea from ammonia. This established for

the ﬁrst time that cyclic, not linear, pathways could play

an important role in central metabolic processes. The

process of this discovery is the subject of a detailed

history dissecting the inventive processes entailed.

One of the great unsolved problems was to determine

the reactions whereby foodstuffs undergo combustion to

produce CO

and water. It was known that for glucose

to undergo these reactions, it must ﬁrst be broken down

to pyruvate in the glycolytic pathway elucidated by Otto

Meyerhof. Although in retrospect, it seems logical to

assume that the idea of metabolic cycles would have

been foremost in Krebs’ mind when he began his

FIGURE 1 Hans Krebs at age 68 in his laboratory at Oxford standing next to his beloved Warburg apparatus, which he learned to use in the

laboratory of Otto Warburg and which was the major instrument used in his discovery of both the urea and the citric acid cycles. The square box in

the background is a power supply for a Zeiss PMQ spectrophotomer. The spectrophotometer was ﬁrst divised by Otto Warburg who also ﬁrst

discovered pyridine nucleotide and observed their spectral shift during oxidation/reduction. This spectral change formed the basis of many of the

assays Krebs undertook in his later years. With these simple instruments and a devoted staff he worked happily in this building at the Radcliffe

Inﬁrmary until he was 81, when he died in a hospital ward in the same building in 1981 after a 2 week illness.

TRICARBOXYLIC ACID CYCLE 257

investigation into the oxidative combustion of food-

stuffs, a careful analysis of his writing and notebooks

gives no support for this idea. Rather his formulation of

the TCA cycle was the result of the empirical methods

learned in Warburg’s lab. From Albert Szent-Gyorgyi, he

had pigeon breast muscle minces which preserved intact,

the then unknown mitochondria containing the enzymes

of the TCA cycle, and could oxidatively decompose

glucose to CO

. From Warburg he had a manometer

with which he could measure rates of CO

production.

Again, he added possible intermediates and observed

whether the rates of reactions increased. As he put it in

his autobiography:

One way of tackling the problem of the intermediate

steps of the combustion process is to test which

substances, apart from carbohydrate and fat, burn most

readily. The logic is that if a substance is an intermediate

then it must readily undergo combustion: if it proves to

be non-combustible, it cannot be an intermediate.

In two sentences was his simple plan: the same as he

had used in elucidating the urea cycle. There was no

grand plan. In choosing the intermediates to be added,

he was guided by others working on the same general

problem. Earlier Szent-Gyorgyi had shown that addition

of small amounts of succinate, fumarate, malate, or

oxaloacetate could catalytically increase the rate of

oxidation by pigeon breast muscle minces. Szent-

Gyorgyi interpreted his ﬁndings as indicating that

these compounds were not intermediates in a pathway,

but rather that they served as hydrogen carriers

transporting the H

and electrons from foodstuffs to

cytochromes. By 1937, experiments by Martius and

Knoop, Krebs’ teacher at Freiburg, had shown in liver

that citrate was converted to aconitate, then isocitrate

and

-ketoglutarate, which in turn was known to be

converted to succinate. It was also known from

Thunberg’s work that malonate could inhibit the

conversion of succinate to fumarate. This presented a

way for Krebs to separate the reactions of Carl Martius

and Franz Knoop from those of Albert Szent-Gyorgyi.

Krebs then hit on the crucial question:

So I asked myself whether perhaps oxaloacetate,

together with a substance derived from foodstuffs,

might combine to form citrate again, after the manner

of a cycle.

Krebs began his “Results” section on the discovery of

the tricarboxcylic acid cycle with the experiments or

data showing the catalytic effects of citrate on respir-

ation in pigeon breast minces. His experimental results

conﬁrmed his hypothesis. In his mind this cycle could

explain the complete combustion of foodstuffs to CO

and water.

Into the Wilderness and Back Again

After his triumph in discovering the urea cycle in 1932

and having been invited by Max Plank to discuss his

ﬁndings, in the same year, on 19 June 1933, he was

forced into exile from his own homeland, to which he

and his father were devoted and whose army he had

served, for reasons of racial identity which he had

rejected. He found refuge in Hopkins Biochemistry

Laboratory at Cambridge, dependent upon the kindness

of strangers.

Triumph would again be followed by rejection, after

Krebs formulation of the TCA cycle. This time, however,

the rejection would not be by the Nazi’s who took over

his homeland, but rejection would come from his

scientiﬁc colleagues. A revolutionary theory always has

its critics. Krebs’ tricarboxcylic acid cycle, published in

1937 after being rejected by Nature, was no exception.

By 1940, Earl Evans, who had been trained in Krebs’ lab

at Shefﬁeld, and Harland Wood, one of America’s most

distinguished biochemists then working at Iowa State,

produced evidence that when C-labeled CO

con-

densed with pyruvate to form oxaloacetate it yielded

-ketoglutarate with the entire radioactivity conﬁned to

the carboxyl group adjacent to the carbonyl group.

Wood suggested that this radioactive evidence was

compatible with the condensation of pyruvate with

oxaloacetate forming aconitate, then isocitrate and

ﬁnally a-ketoglutarate at a rapid rate. If the formation

of citrate from aconitate were only a slow side reaction,

then this would account for the radioactive ﬁndings. For

7 years this reasoning was accepted and Krebs changed

the name of his cycle from the more euphonious “citric

acid cycle” (see Figure 2) to the more cumbersome TCA

cycle, which it bears today to accommodate the

generally accepted view in the biochemical community.

Krebs conﬁned his work to practical nutritional

problems for wartime Britain, which was literally

starving from the depredations of Nazi submarine

attacks. Then in 1948, Alexander Ogston published a

short paper in Nature pointing out that the “three-

point” attachment of the symmetrical citrate molecule

to the citrate synthase enzyme would confer optical

properties to the product of that chemically symmetrical

molecule. The “citric acid cycle” was back in business as

Krebs pointed out in his Harvey Lecture delivered on 17

March 1949. The ﬁnal conﬁrmation of the validity of

Krebs’ original postulate of the “citric acid cycle” came

from Fritz Lipmann’s study of the acetylation of

sulfanilamides where he identiﬁed the so-called “active

acetate” as acetyl coenzyme A. Soon thereafter, Stern,

Ochoa, and Lynen showed that the intermediate through

which pyruvate enters the TCA cycle was acetyl CoA

which then combines with oxaloacetate to form citrate,

conﬁrming Krebs’ original formulation of the cycle.

258

TRICARBOXYLIC ACID CYCLE

These workers also demonstrated that not only carbo-

hydrate, but also fats and ketone bodies form acetyl

CoA. This established that the Krebs citric acid cycle not

only accounted for the complete conversion of carbo-

hydrate to CO

and water, but that this cycle was the

terminal pathway of the degradation of all foodstuffs,

including fats and amino acids (see Figure 3). It may

have been with some irony that Krebs entitled his Nobel

prize speech of 11 December 1953, “the citric acid

cycle” rather than the “TCA cycle” as it was called after

the radioactive data were mistakenly interpreted to

indicate that aconitate, and not citrate, was the

condensation product of carbohydrate degradation.

Subsequently, students of biochemistry would certainly

have found the original name less fearsome than the

“tricarboxcylic acid cycle,” a name that evolved from

erroneous interpretation of experimental results and was

perpetuated by Krebs inherent modesty.

The TCA Cycle Today

It is now accepted that the TCA cycle is not only the

metabolic pathway which accounts for the complete

combustion of the product of glycolysis, pyruvate, to

and H

O that is the pathway which accounts for

the complete combustion of all foodstuffs, carbo-

hydrates, fats, and amino acids in heterotrophic organ-

isms. Much about the control of ﬂux through the TCA

cycle remains unknown even today. One persistent

question is how cyclic pathways appear to defeat the

laws of thermodynamics. It is easy to understand how a

linear metabolic pathway, such as glycolysis, uses the

chemical energy of the reactants to proceed from A to E.

It is not so clear, however, how a pathway which goes

from A to A accomplishes this feat in an apparent

violation of thermodynamic imperatives.

The eight enzymes catalyzing the reactions of the

TCA cycle and their equilibrium constants are listed in

Table I.

The overall reaction of the TCA cycle is therefore:

AcetylCoA þ 2H

O þ 3NAD

þ coenzymeQ þ GDP þ Pi

! 2CO

þ CoA þ 2H

þ 3NADH

þ coenzymeQH

þ GTP

with sum standard free energies, DG

at pH 7 of

2 11.7 kcal mol

. The sum of standard free energies

of the reactions of the cycle is negative. While the sum of

the standard free energies of the reactions of the cycle

are negative in the direction of the cycle written, there is

no complete information on the actual DG

of the cycle

in vivo. The actual DG

of a reaction is of course, a sum

of the standard free energy, DG

, and a term represent-

ing the actual ratio of the concentration of products

over reactants.

Without knowledge of the ratio of concentrations in

mitochondria of the products over the reactants of the

various eight steps of the TCA cycle, no deﬁnitive

statement about the reversibility of any step can be

made, although most biochemical textbooks assign one

or another TCA cycle as “irreversible.”

The synthesis of citrate from acetyl CoA and

oxaloacetate, by citrate synthase, has the largest

standard free energy of any reaction within the cycle

and might therefore qualify for the irreversible desig-

nation. However, the distinguished biochemist, Paul

Srere, who spent much of his career in biochemistry

studying the kinetics of citrate enzymes, argued force-

fully that the citrate synthase reaction achieved near

equilibrium in vivo. Krebs himself argued that both the

NAD- and NADP-dependent mitochondrial isocitrate

dehydrogenases were in near equilibrium with the free

mitochondrial [NAD

]/[NADH] pool. A near-equili-

brium reaction is hardly compatible with irreversibility.

''triose''

Citric acid

(Wagner-Jauregg and Rauen)

(Martius and Knoop)

(Green)

Iso-citric acid

Oxalo-succinic acid

Succinic acid

Fumaric acid

l-Malic acid

Oxalo-acetic acid

a-Ketoglutaric acid

FIGURE 2 The original schematic of the citric acid cycle as it appeared in an article by Krebs and Johnson in 1937, after having been rejected by

the editor of Nature. Krebs attempted to deﬁne the “reversible” and “irreversible” reactions of the cycle as he knew them at the time, but which

would not be accepted as correct today. The nature of the “triose” combining with oxaloacetate was not known until the identiﬁcation of acetyl

CoA by Lipmann and Ochoa in the 1950s. The essential nature of the cycle as drawn in 1937 is however, correct to this day.

TRICARBOXYLIC ACID CYCLE 259

The reaction catalyzed by the

-ketoglutarate dehydro-

genase multienzyme complex could well be irreversible,

but speciﬁc knowledge allowing one to come to a ﬁrm

conclusion is lacking.

What is clear is that the disposal, of the reducing

power produced in the reactions of the TCA cycle,

mainly in the mitochondrial electron transport system

critically affects the extent and possibly the direction of

the reactions of the cycle. Within the last decade, it has

been established that the TCA cycle exists in the earliest

surviving life forms, the archebacteria which developed

when Earth was covered in a reducing atmosphere of

and NH

and no O

. Archebacteria contain a

TCA cycle, which was proposed to serve as a primary

anaplerotic source of metabolites and amino acids, not

as the primary pathway for the degradation of foodstuffs

in heterotrophs. When life forms were developing, in the

absence of O

, the direction of the TCA cycle would

have been reversed.

Whether one accepts this speculation or not, it

emphasizes the intimate relationship between the TCA

cycle and the production of reducing power in the form of

reduced pyridine nucleotide, which serves as the primary

substrate for the electron transport system. When

formulated in 1937, Krebs was primarily concerned

with the stoichiometry of reactions accounting for the

transformation of foodstuffs into CO

. In 1936, Otto

Warburg had just discovered NADP in red cells, so the

central role of these essential cofactors was not

appreciated until much later when Krebs played a crucial

role in deﬁning the redox potential of the mitochondrial

[NAD

]/[NADH] ratio using principles ﬁrst deﬁned

Holzer, Lynen, Bucher, and Klingenberg in deﬁning the

free cytoplasmic [NAD

]/[NADH]. Krebs and his

LDH

PDH

Citrate synthase

MDH

Fumarase

Succ DH

Succinyl CoA ligase

3-oxoaciid CoA transferase

Acetyl CoA transferase

aKDH

ICDH

Aconitase

GLDH

HBDH

Lactate

−

Pyruvate

−

Acetyl CoA

Citrate

−

Aconitate

−

Isocitrate

−

a-Ketoglutarate

−

Succinyl CoA

−

Succinate

−

Fumarate

−

Malate

−

Oxaloacetate

−

Acetoacetate

−

Acetoacetil CoA

CoA

D-b-hydroxybutyrate

−

Glutamate

−

NAD

NADHc+H

FAD

FADH

NADHm

NAD

CoA

NAD(P)

NAD(P)H

NADHm

NAD

+ H

NADHm

NAD

+ H

FAD

FADH

CoA

GTP GDP

CoQH

CoQ

FAD

FADH

NADHm

NAD

Histidine

Proline

Arginine

Alanine

Serine

Cysteine

Fatty acids

Leucine

Isoleucine

Aconitase

Asparatate

Tyrosine

Phenylalanine

FIGURE 3 The TCA cycle is now recognized as the central pathway for the complete degradation of the major foodstuffs including carbohydrate,

lipid, and proteins. It also captures the reducing equivalents which are then transferred to the electron transport system of mitochondria where the

energy of their redox reactions generate the mitochondrial proton gradient used to power ATP synthesis.

260 TRICARBOXYLIC ACID CYCLE

colleagues then went on to show that the redox potentials

of all of pyridine nucleotides were related to one another

in a coherent manner. The free cytosolic NAD couple was

about 2 0.19 V, and hence could accept reducing

equivalents from glycolysis, the free mitochondrial

NAD couple was a more negative 2 0.28 V, providing

the energy necessary for mitochondrial ATP generation,

while the free cytosolic NADP system was the most

negative at 2 0.41 V, required for the completion of the

reductive synthesis of fats. While it was recognized at the

time that the pyridine nucleotides were related to the free

[ATP]/[ADP][Pi] ratio, subtleties in the effects of free

[Mg

2þ

] complicated this relationship and it took another

10 years to accurately determine the equilibrium

constants required to quantitatively deﬁne that

relationship.

In addition to accounting for the chemical decompo-

sition of foodstuffs, it is now recognized that the TCA

cycle produces reducing power in the form of three

reduced pyridine nucleotides and one reduced coenzyme

Q within the mitochondria. Each of these cofactors

contains two electrons. Those contained in NADH feed

into the mitochondrial electron transport system at

complex I, the NADH dehydrogenase multienzyme

complex. The reducing equivalents contained in reduced

coenzyme Q, generated in the conversion of succinate to

fumarate feed into the electron transport chain at a site

with a higher redox potential than that of the

mitochondrial [NAD

]/[NADH] couple. Approxi-

mately four protons are ejected from mitochondria at

each site creating a DG [H

]cytosol/mitochondria,

which provides the energy approximately equivalent to

that of the DG

of ATP hydrolysis in a near equilibrium

reaction catalyzed by the F1 ATPase.

It was originally thought that the TCA cycle was so

fundamental that any defects in the cycle would be fatal.

It is now recognized that because of its central role, not

only in the degradation of food stuffs, the TCA cycle

plays a central role in generating the redox potentials not

only of the mitochondrial NAD couple but also of the

mitochondrial Q couple. The difference in the redox

potential of these two couple, in turn, sets the magnitude

of the energy of the proton gradient between

the mitochondrial and cytosolic phases of the cell and

thus determines the energy realized when the ATP

formed by the mitochondria is hydrolyzed. The amount

of realizable energy formed is a function of the

amount and type of the substrate being utilized by

mitochondrial TCA cycle.