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Anaplerotic Sequences in Microbial Metabolism.

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Anaplerotic Sequences in Microbial Metabolism [lJ
BY PROF. DR. H. L. KORNBERG
DEPARTMENT O F BIOCHEMISTRY, UNIVERSITY OF LEICESTER (ENGLAND)
In addition to catabolic and anabolic pathways, which enable nutrients to enter and to be
withdrawn from the central routes of metabolism, enzymic sequences operate to ensure
that intermediates, removed from these central routes in the course of biosynthesis, are
replenished. These obligatory path ways of replenishment are generically designafed “anaplerotic sequences”. The nature of these sequences, operative in micro-organisms growing
on C3- and C2-compounds, is discussed in outline, and the mode of regulation of one of
them (the glyoxylate cycle) is examined in detail.
Metabolic routes are sequences of chemical steps,
catalysed by enzymes, which enable living organisms to
obtain from their food both the ingredients required for
the synthesis of cell constituents and the energy necessary to effect such and other endergonic processes. The
routes whereby this is achieved are generally divided into
three distinct types. C a t a b o l i c routes serve primarily
to promote the entry of individual nutrients into the
c e n t r a l , energy-yielding pathways of metabolism (the
glycolytic sequence and tricarboxylic acid cycle) ;
a n a b o l i c routes utilize the intermediates of these
pathways as precursors of the organism’s own macromolecules. The c e n t r a l pathways thus play a dual role,
and the intermediates which lie upon them are indispensable to the provision both of energy and of the carbon
skeletons of the multitudinous components of the cell:
by virtue of this dual function, these pathways have been
termed a m p h i b o l i c [2].
However, these three types of metabolic routes are not
sufficient to account for the growth of micro-organisms,
provided with a single compound as sole carbon source,
when the catabolism of that compound cannot directly
yield an intermediate of the tricarboxylic acid cycle:
some route ancillary to that cycle must operate to ensure
that the intermediates of the cycle, which are drained
away during growth, are replenished. It is the main
purpose of this article to discuss the nature of these
obligatory routes of replenishment, which may be
generically described as a n a p l e r o t i c s e q u e n c e s [3].
atoms to form acetyl-coenzyme A, which enters the
cycle after condensation with oxaloacetate. Each turn of
the cycle then results in the loss of a further two carbon
atoms as carbon dioxide and regeneration of the oxaloacetate necessary to accept a further C2-unit. In the
growing cell, however, the operation of anabolic routes
for the synthesis of, for example, porphyrins, and
amino acids of the glutamate and aspartate “families”,
removes the intermediates required for this necessary
regeneration of oxaloacetate: unless an anaplerotic
reaction also occurs, which enables this loss of tricarboxylic acid cycle intermediates to be made good, both
energy provision and biosynthesis must cease.
In the example cited, this anaplerotic function is borne
by carboxylation reactions (Fig. 1). In Pseudomonas [4],
oxaloacetate may be formed by the direct carboxylation
of pyruvate (Reaction a) but this reaction does not
appear to occur in the Enterobacteriaceae [ 5 ] . Instead,
three other enzymes theoretically capable of effecting
the net formation of C4-acids from the carboxylation of
CH3-CO-CO2H
+ ATP + C 0 2 +
f ADP
HOzC-CH2-CO-COzH
+ H3P04
(a)
Carbohydrate
J
Phospho-,
enolpyruvate
\
+
Pyruvate
1. Anaplerotic COz-Fixation
The catabolism of hexoses, glycerol, or other precursors
of pyruvate yields C3-compounds which, in aerobic
organisms, are totally combusted via the tricarboxylic
acid cycle. Pyruvate initially loses one of its carbon
[I] Substance of a plenary lecture, delivered o n October 22nd,
1964, a t the Herbsttagung der Gesellschaft fur Physiologische
Chemie, Koln (Germany).
121 B. D.Davis, Cold Spring Harbor Sympos. quantltat. Biol. 26,
1 (1961).
[3] I am indebted to Professor A. Wusserstein who from the
wealth of his classical erudition concluded that, if “anaplerotic”
did not exist, it ought to be invented. Subsequent investigation
showed the word to be respectably enshrined both in the Oxford
English Dictionary and in the vocabulary of Pathology.
558
lAL471/
-4%
Porphyrins
Fig. 1. Anaplerotic fixation of carbon dioxide. The net formation of
C4-compounds through the carboxylation of C,-acids (indicated by
black arrow) is necessary for replenishment of tricarboxylic acid cycle
intermediates, removed in the course of growth.
[4] W. Seubert and
V. Remberger, Biochem. Z. 334, 401 (1961).
[ 5 ] J. M. Ashworth, H . L. Kornberg, and R. L. Ward, Biochem.
J. 94, 28P (1965).
Angew. Chem. internat. Edit. J Voi. 4 (1965) 1 No. 7
I Acetate 1
C3-compounds have been found to be present: these are
(Reaction b) phosphoenolpyruvate carboxylase [ 6 ] ,
CHz=C(COzH)-OPO3Hz
+ C 0 2 + HzO
+ H02C-CO -CH2 -COzH
+ H3P04
(b)
(Reaction c) “malic enzyme” [7],
CH, -CO--C02H
;t
+ NADPH2 -t C 0 2
HOzC -CHOH-CH2 -CO2H -t NADP
(c)
and (Reaction d) the ATP-linked oxaloacetate decarboxylase [S]
CH2=C(C02H)--OP03H2
~2
+ ADP + C 0 2 + H 2 0
+ ATP
HOzC-CO-CH2-COzH
(d)
It was possible to distinguish the enzyme bearing the
anaplerotic function by studies [ 5 ] with a mutant of
Escherichia coli which, though unable to grow on glucose
plus ammonia, grew readily if this medium was supplemented with any intermediate of the tricarboxylic acid
cycle able to penetrate the cell. Washed suspensions of
this mutant oxidized pyruvate only to the level of acetate,
which accumulated; however, pyruvate was totally
combusted if catalytic amounts of r-malate were also
provided.
Ultrasonic extracts of t h e mutant contained a s much of
enzymes for reactions (c) an d (d) as did similar extracts of
t he parent, wild-type organism, b u t differed from them in
being devoid of the ability t o catalyse reaction (b). This
showed tha t t h e enzyme necessary for t h e net formation of
Cs-compounds was a (virtually irreversible) phosphoenolpyruvate carboxylase, an d suggested th at anaplerotic enzymes a re distinguishable f r o m others which, while effecting
similar reactions. serve a different function in th e cell.
Fig. 2. The tricarboxylic acid cycle and the glyoxylate cycle. The nnaplerotic nature of the latter is indicated by heavy arrows.
succinate and glyoxylate [ 12-14], the second, malate
synthase (Reaction f) catalyses the condensation of the
glyoxylate thus formed with acetyl-coenzyme A to yield
malate [15]. Together with the steps of the tricarboxylic
acid cycle (which, of course, provides the energy for
growth on acetate) leading from malate to isocitrate,
this anaplerotic route serves to maintain the supply of
tricarboxylic acid cycle intermediates as they are removed
for biosyntheses. The overall effect of this sequence
(Reaction g), which has been termed the glyoxylate
cycle [16,17], is formally identical with the often-postulated “Thunberg condensation” [ 181 though the mechanism is quite different.
acetyl-coenzyme A
+ oxaloacetate + H2O
2. Anaplerotic Sequences in Acetate Metabolism
isocitrate
+ HzO
--f
acetyl-coenzyme A + glyoxylate
malate
Although mutants of E. coli devoid of phosphoenolpyruvate carboxylase are unable to grow on pyruvate or
precursors thereof, such mutants grow readily on acetate
as sole carbon source [9]. This shows that some other
anaplerotic sequence operates under these conditions to
effect the net formation of tricarboxylic acid cycle intermediates as these are removed in the course of growth.
This sequence has been shown [lo] to consist of two
enzymes, the concerted action of which serves to bypass the decarboxylative steps of the tricarboxylic acid
cycle and to lead to a net increase in the organic carbon
content of the cell (Fig. 2).
--f
+
+
citrate
2 acetyl-coenzyme A
+
1/2 0 2
-+
+ l/2 0 2
+
citrate
+ coenzyme A
isocitrate
+ glyoxylate (e)
+ coenzyme A (f)
oxaloacetate + H z 0
succinate + 2 coenzyme A
+ Hz0 (g)
succinate
malate
3. Anaplerotic Sequences in Glyoxylate Metabolism
The first of these enzymes, isocitrate lyase (Reaction e),
catalyses the aldol cleavage of threu-D-isocitrate [l 11 to
When glyoxylate, or precursors of glyoxylate, serve as
carbon source for growth, the tricarboxylic acid cycle
still serves to supply biosynthetic intermediates but does
not act as the terminal respiratory pathway. Instead,
glyoxylate is totally combusted via a dicarboxylic acid
cycle (Fig. 3), which comprises some but not all of the
component reactions of the tricarboxylic acid cycle.
[61 R . S. Bundurski and C . M . Greiner, J. biol. Chemistry 204,
781 (1953).
[7] S. Ochoa, A . H . Melrler, and A . Kornberg, J. biol. Chemistry
174, 979 (1948).
[8] M . F. Utter and K. Kurahashi, J. biol. Chemistry 207, 787
(1954).
[91 J . M . Ashworth and H. L. Kornberg, Biochim. biophysica
Acta 73, 519 (1963).
[ 101 For review, see H. L. Kornberg, Annual Rev. Microbiol. 13,
49 (1959); H . L. Kornberg and S . R . Elsden, Advances in Enzymol.
23, 401 (1961).
[ I l l H. B. Vickerj, J. biol. Chemistry 237, 1739 (1962).
[I21 J . J . R . Campbell, R . A . Smith, and B. A . Eagles, Biochim.
biophysica Acta J I , 594 (1953).
1131 J. A . Olson, Nature (London) 174, 695 (1954).
[ 141 R. A . Smith and I. C . Gunsalus, J . Amer. chem. SOC.76,5002
(1954).
[15] D.T. 0. Wong and S . J. AjC, J. Amer. chem. SOC.78, 3230
(1956).
1161 H. L. Kornberg and N . B. Madsen, Biochim. biophysica
Acta 24, 651 (1957); Biochem. J. 68, 549 (1958).
1171 H . L. Kornberg and H . A . Krebs, Nature (London) 199, 988
(1957).
[18] T.Thunberg, Skand. Arch. Physiol. 40, I (1920).
.. -
Angew. Chem. internat. Edit.
/
Vol. 4 (Z965)
1 No. 7
..
~~
559
Glyoxylate
Tartronic semialdehyde
\ /-2H
'c
lGlyoxylate]
I
Glycerate
A
I
Acetyl-coenzyme A Malate
2H
i
1P
!-"
Oxaloacetate
ATP?CO:
,., .
Cell components
Fig. 3. The dicarhoxylic acid cycle for the oxidation of glyoxylate (light
arrows) and the glycerate pathway (heavy arrows) which maintains the
necessary levels of dicarboxylic acid cycle intermediates.
In this cycle,malate synthetase(Reactionf)plays a respiratory
role. The initial condensation of glyoxylate with acetylcoenzyme A, catalysed by this enzyme, yields malate, which
is further oxidized via oxaloacetate and pyruvate t o regenerate
the acetyl-coenzyme A acceptor molecule [19]. The removal
of precursors for cellular syntheses from this cycle (and all
the intermediates of the cycle can act as such) would cause
oxidation t o cease: an anaplerotic sequence must therefore
operate to effect the net formation of such intermediates from
glyoxylate. This sequence has been shown [20] to consist of
three enzymes: glyoxylate carboligase [21], which catalyses
the formation of tartronic semialdehyde from the condensation of 2 moles of glyoxylate, with elimination of 1 mole of
carbon dioxide (Reaction h) ; tartronic semialdehyde reductase 1221 which effects the reduction to glycerate of the
tartronic semialdehyde thus formed (Reaction i); and
glycerate kinase [ 2 3 ] , which enables the glycerate t o be
transformed into phosphoglycerate (Reaction k), an intermediate of an amphibolic pathway. The overall reaction (1)
is that of the glycerate pathway [24] (Fig. 3).
2 glyoxylate
+ 2H
+ ATP
2 glyoxylate + ATP + 2 H
tartronic semialdehyde
glycerate
+ tartronic semialdehyde
+ C02
(h)
+
glycerate
(0
--f
phosphoglycerate
+ ADP
phosphoglycerate + ADP + C02
(k)
--f
(1)
The phosphoglycerate thus produced is metabolized t o
acetyl-coenzyme A and, by condensation with a further
molecule of the growth substrate, i.e. of glyoxylate, enables
the dicarboxylic acid cycle to be replenished.
4. Quantitative Aspects of Anaplerosis
The observation that cell-free extracts of microorganisms catalyse a variety of different chemical reactions is, in itself, not sufficient to show that any
__ ____
[191 H. L. Kornbrrg and J. R . Sadler, Nature (London) 185, 153
(1960); Biochern. J. 81, 503 (1961).
[20] H. L . Kornberg and A. M. Gotto, Nature (London) 183,
1791 (1959); Biochem. J. 78, 69 (1961).
[21] G. Krakow, S. S. Barkulis. and J . A. Hayashi, J. Bacteriol.
81, 509 (1961).
[22] A. M . Gotto and H. L. Kornberg, Biochem. J. 81, 273 (1961).
[23] C. C. Doughtyand J. A. Hayashi, Abstr. 6th Internat. Congr.
Biochem. 6, 507 (1964).
I241 H. L. Kornberg, Cold Spring Harbor Symp. quantitat. Biol.
26, 257 (1961).
560
particular sequence of such reactions plays an important
role in the economy of the cell as an anaplerotic pathway.
Such evidence can, however, be readily obtained with
auxotrophic mutants. It has already been mentioned
that mutants of E. coli devoid of phosphoenolpyruvate
carboxylase (Reaction b) are unable to grow on glucose,
glycerol, or C3-acids, though they readily grow on these
compounds when their media are supplemented with
any intermediate of the tricarboxylic acid cycle to which
the cells are permeable; such mutants also grow on
acetate or glycolate as sole carbon sources, since the
anaplerotic sequences nesessary under these conditions
(Figs. 2 and 3) do not involve the carboxylation of phosphoenolpyruvate. Similarly, mutants of E. coli devoid of
isocitrate lyase (Reaction e) do not grow on acetate but
grow readily on glucose, intermediates of the tricarboxylic acid cycle or glycolate; revertants of such mutants,
which regained the ability to grow on acetate, were found
concomitantly to have regained isocitrate lyase activity
[25]. It is further of interest that E. coli has been found
[26] to contain two malate synthases with different
properties: this may reflect the catabolic role of this
enzyme in the dicarboxylic acid cycle and its anaplerotic
function in the glyoxylate cycle and glycerate pathway.
A second type of evidence for the intracellular operation
as metabolic pathways of the enzymic sequences here
described comes from measurement of the amounts of
pacemaker enzymes [27] present in extracts of E. coli
grown on appropriate carbon sources (Table 1): the
activities of these enzymes are more than adequate to
account for the replenishment of tricarboxylic acid cycle
intermediates during growth. It may thus be concluded
that the various anaplerotic sequences are both necessary
and sufficient for growth.
Table 1. Levels of some anaplerotic enzymes in extracts of E. coli W at
3 0 "C (adapted From 1241).
1 Average specific activity [Vmoles/mg protein/hour] of
~~
Growth
substrate
Lactate
Acetate
Glycolate
II
Isocitrate
lvase
(Reacn. e)
0.5
II
Malate
svnthetase
(Reacn. f)
I
1
Glyoxylate
carbolinase
(Reacn. h )
I
1
1 1 I
0.4
3:
I
2;
Tartronic semialdehyde
reductase
(Reacn. i)
4
:7
5. Regulation of Anaplerotic Sequences
It is further apparent from the data presented in Table 1
that the pacemaker enzymes of anaplerotic sequences
are under metabolic control: the appropriate enzyme is
found to be abundant in extracts of cells only when the
activity of that enzyme is necessary for growth on the
relevant carbon source. This phenomenon, whereby
micro-organisms apparently select [24] the correct
[25] J . M . Ashworth and H . L. Kornberg, Biochim. biophysica
Acta 89, 383 (1964).
[26] P. Falmagne, E. Vanderwinkel, and J. M . Wiame, Arch. internat. Physiol. Biochim. 71, 813 (1963).
1271 H. A . Krebs and H. L. Kornberg, Ergebn. Physiol., biol.
Chem. exp. Pharrnakol. 49, 212 (1957).
Angew. Chem. internat. Edit. J Vol. 4 (1965) / No. 7
metabolic route from the range of routes which they are
genetically capable of using, is illustrated by the changes
in the ratio of malate synthase to citrate synthase as
lactate-grown E. coli continue to grow on lactate, or
adapt to growth on acetate or glycolate (Fig. 4).
I
A
Y-o-o-oTo
!
a) Influence of Growth Substrates on Isocitrate
Lyase Levels
The specific activity of isocitrate lyase found in extracts
of acetate-grown E. coli, strain W, was 15.3 pmoles of
glyoxylate formedlmg of protein/hour, which was considerably higher than after growth on other carbon
sources (Fig. 5).
Glucose L0.321
I /
Olycerol U . 2 1 4
m
q
d
k
Proirne [Ull
Fig. 4. Variation with growth of the ratio of malate synthase to citrate
synthase in lactate-grown E. coli W continuing to grow on lactate
( Y ) or adapting to growth on acetate ( 0 ) or on glycolate (0).
Citrate
a-Oxoglutarate [1,21--
!
Glutamate 13.81
Ordinate: Activity of malate synthetase/activity of citrate synthetase.
Abscissa: Cell density [mg dry ueight!mll.
In all of these circumstances, the correct ratio of
enzymic activity appears to be achieved before any major
increase in the number of cells has occurred: these
ratios are then maintained throughout subsequent
growth [28]. This suggests that the differential rate of
synthesis of pacemaker enzymes of anaplerotic sequences
is regulated by variations in the intracellular levels of
specific metabolites.
6. “Coarse Control” of the Glyoxylate Cycle
Although the “selection” of a particular metabolic route
is a general phenomenon (and is by no means confined
to anaplerotic sequences [29]), the mechanisms whereby
anaplerotic sequences are regulated have been studied
in detail for only one such sequence: the glyoxylate
cycle. This study [30] has concentrated largely on
analysis of the factors which regulate the formation of
isocitrate lyase in E. coli W , E. coli M 22-64 (a mutant
of E. coli W , devoid of citrate synthase [31]), E. coli
M 191-6 (a mutant of E. coli W , devoid of pyruvate
oxidase [32]), E. coli Bm [33] (a mutant of E. coli B,
devoid o f phosphoenolpyruvate carboxylase [9]), and
Achroniobacter d-15 [34].
. . ..
.-
[28] J. R. Snder, Ph. D. Thesis, University of Oxford, 1961.
[29] For a recent survey, see H. E. Umbarger, Science (Washington) 145, 674 (1964).
1301 H . L. Kornberg, Colloq. internat. C.N.R.S., Marseille, 1963.
[31] C. Gilvarg and B. D. Davis, J. biol. Chemistry 222, 307
( 1 956).
[32] A . D . Gounaris and L . P . Hager, J. biol. Chemistry 236,
1013 (1961).
I331 A gift from Prof. L. Gorini and Mr. R. E. Ljwch (Harvard
Medical School, Boston, Mass., U.S.A.).
[34] A gift from Dr. R. F. Rosenberger (Hebrew University Hadassah Medical School, Jerusalem, Israel).
Angew. Cltem. internat. Edit. / Vol. 4 (1965)
/ No. 7
Fig. 5. Influence of growth substrates on isocitrate lyase levels in
extracts of E. coli W. The observed specific activity of the enzyme is
given in square brackets after the name of the growth substrate. The
hatched bars indicate the site of the lesions in the mutants used.
However, even the magnitude of these lower specific
activities was influenced by the nature of the growth
substrate, as the values observed ranged from 0.25, for
pyruvate-grown cells, to 4.0, for proline-grown cells. An
indication of the probable nature of the phenomena
underlying these variations is derived from two correlations :
a) The enzyme levels achieved on any carbon source
appear to be related to the place which that carbon
source occupies on the “metabolic map” (Fig. 5). Thus,
growth on pyruvate and on direct precursors of pyruvate
is accompanied by the lowest specific activities of isocitrate lyase, and the interposition of an additional
enzymic step before the growth substrate can enter the
metabolic pathways leading to C3-compounds is accompanied by an increased production of the enzyme.
b) The enzyme levels achieved during growth on any
carbon source (other than acetate) appear to be inversely
related to the growth rate on that carbon source (Fig. 6).
The highest specific activities of isocitrate lyase were found
during growth on substrates such as proline, glutamate, yaminobutyrate, and glycolate, which require the occurrence
of one or more reactions before they can enter the EmbdenMeyerhof pathway or the tricarboxylic acid cycle: growth o n
these substances was slower than on the intermediates of the
main metabolic routes t o which they are transformed.
These observations support the view that the rate of
formation of isocitrate lyase is governed by the intracellular levels of metabolites which act as repressors.
Among the factors determining these levels would be the
rate of entry of the growth substrate into the main
metabolic routes, which would thus influence the rate of
growth of the organism.
56 I
failure of acetate to stimulate the rate of isocitrate lyase
synthesis by M 22-64 argues against acetate (or acetylcoenzyme A) as a direct inducer of this enzyme. These
observations are, however, those expected if the differential rate of isocitrate lyase formation is governed by
the intracellular levels of some repressor substance(s)
metabolically close to oxaIoacetate : in the wild-type
organism, acetate would serve to lower these levels by
the formation of citrate but, in the mutant, would be
unable to act in this manner.
Fig. 6. Relation between mean doubling time and specific activity of
isocitrate lyase in E coli W, grown on the following substances as
sole carbon sources: 1: Proline, 2: glutamate, 3: y-aminobutyrate,
4: glycolate, 5: aspartate, 6: glycero!, 7 : succinate, 8: alanine, 9: fumarate, 10: malate, 1 1 : lactate, 1 2 fructose, 13: glucose, 14: glycerate,
15: pyruvate.
Ordinate: Specific activity of isocitrate lyase.
Abscissa: Mean doubling time.
b) The Role of Acetate in Isocitrate Lyase Induction
The high rate of enzyme synthesis during growth on
acetate appears to be an exception to these two correlations (a) and (b) and might imply that acetate, or
acetyl-coenzyme A, acts as a direct inducer of isocitrate
lyase synthesis. There are several lines of evidence
against this [35].
Identical specific activities of isocitrate lyase were
obtained when E. coli were grown on media containing
the carbon sources listed in Fig. 6 (with the exception of
proline, glutamate, and y-aminobutyrate) at 20 mM
concentration, and in similar media which, in addition,
contained 20mM acetate. This showed that, in the
presence of these substances, acetate did not induce isocitrate lyase formation. Only when proline, glutamate,
or y-aminobutyrate were the growth substrates did the
inclusion of acetate in the growth medium stimulate the
rate of isocitrate lyase synthesis : this “inducer-like’’
effect of acetate [36] which resulted in isocitrate lyase
levels 2-3 times higher than those observed with
organisms grown in the absence of acetate, was obtained
also when ammonium chloride was omitted from the
growth medium and the amino acids thus served as the
sole nitrogen as well as the main carbon sources.
However, whereas growth of the wild-type organism
(E. coli, strain W ) on proline plus acetate, glutamate
plus acetate, or y-aminobutyrate plus acetate, resulted
in higher rates of isocitrate lyase formation than observed
during growth on these amino acids alone, the rates of
enzyme formation in the mutant M22-64 (which lacked
the ability to form citrate from oxaloacetate and acetylcoenzyme A, see Fig. 5 ) were unaffected by the presence
of acetate (Fig. 7).
Since this mutant has been shown to be still capable of
activating acetate to acetyl-coenzyme A [31,191, the
1351 H. L. Kornberg, Biochim. biophysica Acta 73, 517 (1963).
136) R. F. Rosenberger, Biochim. biophysica Acta 64, 168 (1962).
562
1
i
L
+
L
rn
30
9--
Fig. 7. Effect of 20 mM acetate on isozitrate lyase formation by E. coli W
(0) and its mutant M 22-64
( 0 ) during growth on proline. Acetate
was added at the points indicated by arrows.
Ordinate: Specific activity of isocitrate lyase.
Abscissa: Number of bacterial generations.
Further evidence for this view was provided from the
effect of acetate on M 22-64 growing on 20mM
glycolate plus 2 mM glutamate. In the absence of acetate, isocitrate lyase was produced at a specific activity of
2.2, which remained constant over nearly 2 generations.
The addition of 50 mM acetate after this time not only
failed to induce isocitrate lyase formation but caused a
sharp decrease in the specific activity of the enzyme
without causing any decrease in the growth rate. Similarly, when the glycolate/gIutamate-grown mutant was
inoculated into a medium containing these carbon
sources plus 50 mM acetate, the growth of the organisms
was accompanied by an initial decrease in the content
of isocitrate lyase to a new level, which was less than 6 0 %
of that present in cells growing in the absence of acetate
and which was maintained constant during the subsequent growth (Fig. 8).
Since it is known [19] that acetate can enter the metabolic
pathways of this mutant during growth on glycolate, by
combining with glyoxylate to form malate (cf. Fig. 3),
I
10‘
[m
I
I
I
20
10
9-
Fig. 8. Effect of acetate on isocitrate lyase formation by M 22-64
growing on 20 mM glycolate plus 2 mM glutamate (0)or on this medium
plus 50 m M acetate (e) The experiments were begun with cells grown
on glycolate plus glutamate; acetate was added at the points indicated
by arrows.
Ordinate: Specific activity of isocitrate lyase.
Abscissa: Number of generations.
Angew. Chem. internat. Edit./ VoI. 4 (1965)
/ No. 7
these observations are consistent with the formation
from malate of repressor(s) of isocitrate lyase.
c) Possible Identity of the Metabolite Responsible for
Isocitrate Lyase Repression
It was observed that, during growth on glycolate plus
acetate, the specific activity of isocitrate lyase in the
mutant M 191-6 (which could not oxidize pyruvate)
was less than 30% of that present in M 22-64 (Fig. 8)
and less than 15 % of that in the parent wild-type E. coli
(Fig. 5 ) under similar growth conditions. This suggested
that, if a particular metabolite acted as a specific repressor of isocitrate lyase synthesis, that metabolite is likely
to be a C3-compound rather than an intermediate of the
tricarboxylic acid cycle. There are several other lines of
evidence in favor of this hypothesis:
( I ) The acetate-requiring mutant M 191-6 was grown on a
mixture of 20 mM glycolate plus 10 mM acetate. After a n
increase in cell density of approximately 0. I mg dry weight/
mi, samples of the cultures were taken for enzyme assay and
the volumes of liquid removed were replaced by fresh growth
medium. Under thesc conditions, in which the organism grew
at constant rate and remained at relatively constant cell
density, they produced isocitrate lyase at constant though
unusually low specific activity (0.3 [~.moles/mgprotein/hour).
However, when the liquid removed in the course of sampling
was replaced by equivalent volumes of 20 mM glycolate
medium from which acetate had been omitted, the exhaustion
of the acetate in the growth medium led to a progressive
decrease in growth rate and ultimate cessation of growth.
As expected from the known metabolic lesion of this mutant,
the decrease in growth rate was also accompanied by the
accumulation of pyruvate (identified as the 2,4-dinitrophenylhydrazone) in the medium. The specific activity of isocitrate
lyase in successive samples taken from this culture fell
sharply to very low levels, which suggested that the overproduction of catabolic products from glycolate raised the
concentration of metabolites inside the cells to a level at
which isocitrate lyase synthesis was virtually abolished. Since
the mutant was unable to oxidize pyruvate to acetate and
Iicnce was unable to form malate from glyoxylate in the
absence of added acetate, this unusually strong repression of
isocitrate lyase synthesis occurred under conditions characterized by an over-production of C3-compounds without the
prior formation of malate.
( 2 ) The addition of pyruvate to a culture of A4 22-64 growing
on proline as sole carbon source did not decrease the rate of
growth but markedly lowered the rate of isocitrate lyase
formation (Fig. 9). I n this mutant, the addition of pyruvate
could presumably have raised the intracellular levels of C4dicarboxylic acids as well as those of C3-compounds, but
could not have raised those of other intermediates of the
tricarboxylic acid cycle.
20
T
. m
l5
10
I
02
U
L
J
i
04
d
06
d
Fig. 9. Effect of pyruvate on isocitrate lyase formation by M 22-64
( 4 . A ) and by Bm (0,0 ) at 30°C during growth on proline. Pyruvate
to 5 m M was added to the cultures marked with closed symbols at the
points indicated by arrows.
Ordinate: Specific isocitrate layse activity x cell density.
Abscissa: Cell density Ems dry weightlml].
Angew. Chem. internat. Edit.
Vol. 4 (1965) 1 No. 7
( 3 ) The addition of pyruvate to a culture of the mutant Bn7
growing on proline as sole carbon and nitrogen source did
not decrease the rate of growth but markedly lowered the
rate of isocitrate lyase synthesis (Fig. 9). In this mutant,
pyruvate could be oxidized but could not give rise to the net
formation of C4-dicarboxylic acids.
(4) The addition of pyruvate to a culture of Brn growing o n
50 mM acetate plus 5 mM glutamate, added as sole nitrogen
source, did not decrease the rate of growth of the organism
but led to a decrease of over 85 ;(, in the high rate of isocitrate
lyase synthesis; this effect persisted until the added pyruvate
had been used up (Fig. 10). Since glutamate had been added
as sole nitrogen source, and since no a-0x0 acids were
detected in the medium, the amino acid must have been
utilized for growth but without causing the intracellular
accumulation of repressor substances; such substances were,
however, formed from pyruvate, although no net formation of
C4-dicarboxylic acids from pyruvate could occur in this
mutant
3F
E
Y
2a
k
1
Fig. 10. Effect of pyruvate on isocitrate lyase formation by 3 m during
growth on SO mM acetate plus S m M glutamate, in the absence of other
nitiogen sources. Pyruvate to S mM was added to the culture marked
( 0 ) ; the decrease i n the pyruvate content of the medium is indicated by
( x ). The culture marked (0)received no pyruvate.
Ordinates: left:
right:
Abscissa:
Specific activity of isocitrate lyase x cell density.
Pyruvate concentration [mM].
Cell density [mg dry weight/ml].
( 5 ) The addition of pyruvate to a culture of Bm growing o n
50 mM acetate plus 5 mM aspartate, added as sole nitrogen
source, did not decrease the rate of growth but caused a
sharp decrease in the high rate of isocitrate lyase synthesis
(Fig. 11). Again, the amino acid must have been utilized for
growth and must therefore have provided oxaloacetate to
the organism, but at a rate insufficient to lower significantly
the high rate of isocitrate lyase formation : again, the enzyme
was repressed by the addition of pyruvate although, in this
mutant, this C3-compound could not give rise to the net
formation of C.g-compounds.
These findings suggest that, if any single metabolite may
be regarded as a specific repressor of isocitrate lyase, that
metabolite cannot be a C4-dicarboxylic acid (results
with Bm), cannot be another intermediate of the tricarboxylic acid cycle (results with M22-64), and cannot
be an oxidation product of pyruvate (results with
M 191-6): the metabolite must be either pyruvate
itself or some product metabolically close to both
pyruvate and oxaloacetate.
If it can be assumed that the factors which regulate isocitrate lyase synthesis in E. coliare similar to those which
regulate it in other micro-organisms (and data obtained
563
oxaloacetate (via Reaction d), and since growth on
intermediates of the tricarboxylic acid cycle yields only
low levels of isocitrate lyase, it is tempting to conclude
that one of the repressor metabolites responsible for
regulating the synthesis of isocitrate lyase is phosphoenolpyruvate.
E
7
6
It is probable that more than one metabolite exerts a
regulatory function over isocitrate lyase. Evidence for
this view stems from studies with a mutant, BmR, which,
though derived from Bm, differs from it in being able to
grow on glycerol, glucose, or pyruvate. This growth
pattern is, however, not due to a reversion, with consequent restoration of phosphoenolpyruvate carboxylase
activity, but (as has also been reported 1431 for a similar
mutant of E. coli K 12) is the result of a second mutational change affecting a regulator gene for isocitrate
lyase synthesis (Table 2).
5
Ti.
.-
m3
2
1
dFig. 11. Effect of pyruvate on isocitrate lyase formation by Bm during
growth on 50 m M acetate plus 5 mM aspartate, in the absence of other
nitrogen sources. Pyruvate to 5 mM was added to the culture marked
( 0 ) ; the decrease in the pyruvate content of the medium is indicated by
(x). The culture marked (0)received no pyruvate.
Table 2. Effect of growth substrates o n isocitrate lyase levels in
wild-type E. coli and the mutant B m R at 30 “ C .
Ordinates: left: Specific activity of isocitrate lyase x cell density.
right: Pyruvate concentration ImM].
Abscissa: Cell density [mg dry weight/ml].
Growth
substrate
with bacteria [lo], fungi [37,38], algae [39,40], and
protozoa [41] suggest that this assumption is justified),
then pyruvate itself is not likely to act as a repressor
metabolite. Evidence for this belief stems from results
obtained with Achvomobacter d-IS. This organism is
unusual in that cell-free extracts contain isocitrate lyase
of high specific activity (6-8 pmoles/mg protein/hour)
after growth on pyruvate, lactate, or alanine as sole
carbon sources, though, as observed with other organisms, the enzyme is synthesized to only a small extent
during growth on intermediates of the tricarboxylic acid
cycle. A second unusual feature of Achromobacter d-15
is that it is unable to grow on glucose, glycerol, or
glycolate; this disability may be ascribed [42] to the
absence of pyruvate kinase (Reaction m) activity, with
Glucose
Lactate
Malate
Succinate
Acetate
CI ,-CO-C02H
+ ATP + CH2=C(C02H)O-P03H2 + ADP
Wild-type E. coir
(m)
consequent inability to gain entry into the tricarboxylic
acid cycle for catabolites which must traverse this essential step of the Embden-Meyerhof sequence to do so.
It is thus probable that the high levels of isocitrate lyase
found in extracts of pyruvate-grown Achromubacter
d-15 are similarly symptomatic of the inability of this
organism directly to form phosphoenolpyruvate from
pyruvate[*]; since this substance can still be formed from
[37] J. F. Collins and H. L. Kornberg, Biochem. J. 77,430 (1960).
[38] W . S. Wegener and A . H. Romano, J. Bacteriol. 87, 156
(1 964).
1391 L. C . Harrop and H . L. Kornberg, Biochem. J. 88, 42P
( 1963).
[40] A. G. Calley and D. Lloyd, Biochem. J. 90,483 (1964).
[41] J. F. Hogg and H . L. Kornberg, Biochem. J. 86, 461 (1963).
1421 H . L. Kornberg, J . Dennis, and E. M . Wilson, Biochem. J. 92,
55P (1964).
[*I Note added in proof: It is unlikely that phosphoenolpyruvate can be formed from pyruvate by Reaction (m) since E. coli
contains a PEP-synthase system for this purpose distinct from
pyruvate kinase. - Cf. R. Cooper and H . L. Kornberg, Biochim.
biophysica Acta 104, 618 (1965).
564
Specific activity [v.moles/rng protein/hourl of
isocitrate lyase in
1 Mutant BtnR
0.3
0.5
0.R
10
20
21
I .o
34
15
50
Thus, even during growth on glucose, the differential rate of
isocitrate lyase synthesis is over thirtyfold that observed in
wild-type E. coli, and the necessary net formation of C4-acids
can, in BmR, be effected via the glyoxylate cycle despite the
presence of substrates which strongly repress this enzyme in
wild-type organisms and in Bm. It is, however, remarkable
that extracts of BmR obtained from cells, grown on a
variety of substrates, exhibit a pattern of variation of isocitrate lyase activity with the mean doubling time on that
substrate which is qualitatively similar to that observed with
wild-type cells (cf. Fig. 6 ) : to adapt the famous slogan of
George Orwell‘s “Animal Farm”, it is clear that, although
isocitrate lyase production in BmR is constitutive, it is under
some circumstances more constitutive than under others.
These observations suggest that BmR has escaped from the
repression exerted by one metabolite but remains subject to
repression by another [44].
7. “Fine Control” of the Glyoxylate Cycle
Although the variations in the differential rate of
synthesis of isocitrate lyase described represent one type
of control of the glyoxyIate cycle, such a regulatory
process must be relatively “coarse”. Studies with E. coli,
strain B, and its mutant Bm suggest that, in addition, a
“fine” control of the cycle may be exerted by variations
in the intracellular levels of phosphoenolpyruvate,
which has been found to be a powerful non-competitive
inhibitor of isocitrate lyase [9].
[43] E. Vanderwinkel, P. Liard, I;. Ramos, and J . M . Wianle, Biochem. biophysic. Res. Commun. 12, 157 (1963).
[44] These observations are analogous to the finding that
mutants lacking the i gene for P-galactosidase are still subject to
catabolite repression: see J . Mandelstam, Biochem. J. 82, 489
(1962); W. F. Loomis and B. Magasanik, J . molecular Biol. 8,417
(1964).
Angew. Chem. internat. Edit.
Vol. 4 (1965) 1 No. 7
Although the addition of pyruvate to cultures of E. coli
B growing on acetate, or of Bm growing on acetate in
the presence of glutamate or aspartate, did not decrease
the rate of growth, the addition of this C3-compound to
a culture of the mutant growing on acetate in the
absence of these amino acids speedily arrested its growth,
which was resumed when the added pyruvate had been
utilized (Fig. 12).
neither malate synthase nor citrate synthase was inhibited by 0.1 mM phosphoenolpyruvate, but that isocitrate lyase was inhibited non-competitively, with K,
1.3 x 10-4 M. Other intermediates of the EmbdenMeyerhof pathway (such as 2-phosphoglycerate, 3phosphoglycerate and hexose mono- and di-phosphates)
exerted no inhibitory action at 1 mM concentration.
Similar results have been obtained with the isocitrate
lyase in extracts of E. coli W , Chlorella vulgaris, Brannon
No. 1 strain, and Achromobacter d-15.
Concluding Remarks
Fig 12. Effect of pyruvate a t 30 -C on t h e g r o w t h of E . coli B, ( A ) on
50 m M acetate, a n d o n t h e g r o w t h of its m u t a n t Bm i n media containing
50 m M acetate (o), or 50 m M acetate plus 5 m M glutamate (0).
Sodium pyruvate was a d d e d where indicated by arrows. T h e inset ( A )
records t h e decrease in t h e pyruvate concentration ( m M ) after its
addition 10 Bm growing o n acetate ( 0 ) .
Ordinates: left: Cell density [mg d r y weight/nil].
right: Pyruvate concentration [mM].
Abscissa: T i m e [hours].
It has been the main purpose of this communication to
summarize the evidence for the belief that, for a proper
description of the flow of nutrients associated with the
release of energy and its utilization for biosyntheses, a
special type of metabolic pathway must operate inside
the cell which may be regarded as serving solely to maintain the functioning of the tricarboxylic acid cycle and
other amphibolic routes. It has been proposed to
generically designate this type of ancillary pathway as an
a n a p l e r o t i c s e q u e n c e . Key enzymes of anaplerotic
sequences may be recognized as distinct biological entities, even when other enzymes exist in the cell which
catalyse a closely similar or the same chemical reaction;
their action is necessary for growth on C3- and Cz-compounds to occur (Table 3).
T a b l e 3. Catabolic a n d anaplerotic sequences i n E. coli.
This observation indicated that pyruvate, or a product
of pyruvate metabolism, inhibited the net formation of
C4-compounds from acetate: as an alternative mode of
formation of such compounds, e. g . by carboxylation of
phosphoenolpyruvate, was not possible in this mutant,
its growth ceased. (The failure of pyruvate to inhibit the
growth of Bm on acetate plus aspartate or glutamate is
thus further indication that these amino acids were
indeed utilized as sources of intracellular C4-dicarboxylic acids: cf. Figs. 10 and 11). Since pyruvate was
utilized by 3 m though its growth had ceased, and since
washed suspensions of the acetate-grown mutant readily
oxidized pyruvate, it was evident that pyruvate did not
inhibit a component reaction of the tricarboxylic acid
cycle but probably acted on a key enzyme of the
glyoxylate cycle.
An indication of the mechanism of this effect was provided by the finding that, whereas neither 0.1 mM nor
1 mM pyruvate inhibited significantly the activity of isocitrate 1)ase in cell-free extracts of acetate-grown Bm,
phosphoenolpyruvate was a powerful inhibitor of this
enzyme. At a concentration of 1 x 10-4 M, phosphoenolpyruvate reduced the quantity of glyoxylate formed
from the cleavage of 2 mM isocitrate to 58%, and at
1 x 10-3 M to 11 % of that observed in its absence.
Quantitative spectrophotometric assay [45] showed that
.. _____
I451 G. H . Dixon and H . L. Kortiberg, Biochern. J . 72,3 P (1959).
Angew. Chem. internut. EdiI. / Vol. 4 (1965) / No. 7
Carbon source
Catabolic r o u t e
Anaplerotic sequence
Glucose, &lyceroI
or C,-cornpounds
Acetate
Tricarboxylic
acid cycle I
Tricarboxylic
acid cycle
Dicarboxvlic
acid cycle
Phosphoenolpyruvate
carboxylase
Glyoxylate cycle
Glycolate
Glycerate pathway
Although little is known in detail of the factors which
regulate the operation of the various anaplerotic sequences, studies on the glyoxylate cycle suggest that they
act at least at two levels: via a “fine control” mechanism,
achieved through inhibition of the a c t i v i t y of a pacemaker enzyme, and a “coarse control” mechanism,
achieved through inhibition of the s y n t h e s i s of that
enzyme. The data obtained further suggest that both
modes of ,control may be exerted, at least in part, through
variations in the intracellular levels of the same metabolite - phosphoenolpyruvate. Since phosphoenolpyruvate can be regarded both as the precursor of many
cell components (such as aromatic amino acids, the
pentose moieties of nucleic acids, and other carbohydrates) in their formation from acetate and as the major
product of the glyoxylate cycle, these types of control
are further examples of feedback mechanisms [29],
which enable micro-organisms sensitively and efficiently
to adjust to changes in their environment.
Received: N o v e m b e r 241h, 1964 [A 4471228 IE]
G e r m a n version: Angew. C h e m . 77, 601 (1965)
565
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