Central Metabolic Pathway (White - Chap8)

8
Central Metabolic Pathways
The central metabolic pathways are those is incorporated into an acyl-phosphate

pathways that provide the precursor metabo- (i.e., the oxidation of phosphoglyceralde-
lites to all the other pathways. They are the hyde to 1,3-bisphosphoglycerate). The 1,3-
pathways for the metabolism of carbohydrates bisphosphoglycerate then donates the phos-
and carboxylic acids, such as C4 dicarboxylic phoryl group to ADP in a substrate-level
acids and acetic acid. The major carbohy- phosphorylation. (See Section 7.3.1.)
drate pathways are the Embden-Meyerhof- The fate of the pyruvate that is formed
Parnas pathway (also called the EMP pathway during the catabolism of carbohydrates de-
or glycolysis), the pentose phosphate path- pends on whether the cells are respiring. If
way (PPP), and the Entner-Doudoroff path- the organisms are respiring, then the pyruvate
way (ED). The Entner-Doudoroff pathway that is formed by the carbohydrate catabolic
has been found to be restricted almost entirely pathways is oxidized to acetyl-CoA, which is
to the prokaryotes, being present in gram- subsequently oxidized to carbon dioxide in the
negative and gram-positive bacteria, as well as citric acid cycle. The latter generally operates
archaea. (It has been reported in one amoeba, only during aerobic respiration. (However, see
i.e., Entamoeba histolytica and two fungi, i.e., Section 8.8.4.) If fermentation rather than res-
Aspergillus niger and Penicillium notatum.! )
piration is taking place, then the pyruvate is
The three pathways differ in many ways, but
converted to fermentation end products such
two generalizations can be made:
as alcohols, organic acids, and solvents, rather
1. All three pathways convert glucose to than oxidized in the citric acid cycle. Fermen-
phosphoglyceraldehyde, albeit by different tations are discussed in Chapter 14.
routes. An overview of the carbohydrate catabolic
pathways and their relationship to one
2. The phosphoglyceraldehyde is oxidized to
another and to the citric acid cycle is shown
pyruvate via reactions that are the same in
in Fig. 8.1. Several points can be made
all three pathways.
about this figure. Notice that there are
From an energetic point of view, the reac- three substrate-level phosphorylations, two
tions that convert phosphoglyceraldehyde to during carbohydrate catabolism and one in
pyruvate are extremely important because the citric acid cycle. Furthermore, there are
they generate ATP from inorganic phos- six oxidation reactions, one in glycolysis,
phate and ADP. This is because there is one in the pyruvate dehydrogenase reaction,
an oxidation in which inorganic phosphate and four in the citric acid cycle. These
180
CENTRAL METABOLIC PATHWAYS
NADPH
glucose ~ATP / ATP
~ GiP ~-gIUCO~ ~gluconate
F6P
8 COz_ NADPH
8
Pi
:C IIATP. .~
ATP
I
pentoses
I
1
HzO FBP
~
PGALD
t~-- ATP, NADH
PEP
~--ATP
pyruvate
-- COz.NADH
~
acetyl-CoA
NADH
~oacetate
~ Citrat~
malate [cis-aconitateJ
1
fumarate citric acid
\
isocitrate
FADHz~ cycle
~NADPH
succinate oxalosuccinate
ATP~I-COA a-keto~co
z
/
COz. NADH
NADH+H+
X
B
NAD+ BHz
fermentation
respiration
Fig. 8.1 Relationships between the major carbohydrate pathways and the citric acid cycle.
The pathway from glucose-6-phosphate to pyruvate is the Embden-Meyerhof-Parnas pathway
(glycolysis). The pentose phosphate pathway (PPP) and the Entner-Doudoroff pathway (ED)
branch from 6-phosphogluconate. Both of these pathways intersect with the glycolytic pathway
at phosphoglyceraldehyde. All the carbohydrate pathways produce pyruvate, which is oxidized
to acetyl-CoA. In aerobically growing organisms, the acetyl-Co A is oxidized to C02 in the
citric acid cycle. The electrons from NAD(P)H and FADH2 are transferred to the electron
transport chain in respiring organisms with the formation of ATP. In fermenting cells, the NADH
is reoxidized by an organic acceptor (B) that is generated during catabolism. The citric acid
cycle does not operate as an oxidative pathway during fermentative growth. Abbreviations:
G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-l,6-bisphosphate; PGALD, 3-
phosphoglyceraldehyde; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; ED, Entner-
Doudoroff pathway.
oxidations produce NADH (primarily) and yield depend upon whether the organism is
FADH2. The NADH and FADH2 must be respiring or fermenting. During respiration,
reoxidized in order to regenerate the NAD+ the NADH and FADH2 are reoxidized via
and FAD that are required for the oxida- electron transport with the formation of a !::"p.
tions. The route of reoxidation and the energy (The !::"pis used for ATP synthesis via respira-
181
THE PHYSIOLOGY AND BIOCHEMISTRY OF PROKARYOTES
tory phosphorylation as explained in Chapter Sum: Glucose + 2ADP + 2Pj + 2NAD+

4.) In fermenting cells, most of the NADH + 2 pyruvate + 2ATP + 2NADH + 2H+
is reoxidized in the cytosol by an organic
acceptor, but ATP is not made. (However, The reactions are summarized in Fig. 8.2.
see Note 2.) The different pathways for the The pathway begins with the phosphoryla-
reoxidation of NADH in fermenting bacteria tion of glucose to form glucose-6-phosphate
are discussed in Chapter 14. The student will (reaction 1). The phosphoryl donor is ATP
notice that the citric acid cycle generates a in a reaction catalyzed by hexokinase. The
great deal of NADH and FADHz. The reox- ATP is regenerated from phosphoenolpyru-
idation of the NADH and FADHz requires vate in stage 2. Some bacteria phosphory-
adequate amounts of electron acceptor, such late glucose during transport into the cell
as is provided to respiring organisms. In fact, via the phosphotransferase (PTS) system, in
the oxidative citric acic cycle as illustrated in which case the phosphoryl donor is phos-
Fig. 8.1 is coupled to respiration, and during phoenolpyruvate. (See Section 16.3.4 for a
fermentative growth it becomes modified into discussion of the phosphotransferase system.)
a reductive pathway (Section 8.10). The glucose-6-phosphate (G6P) isomerizes
to fructose-6-phosphate (F6P) in a reaction
catalyzed by the enzyme isomerase (reaction
2). The isomerization is an electron shift where
8.1 Glycolysis
two electrons from the C2 carbon reduce
the C1 aldehyde of the glucose-6-phosphate
It is best to think of glycolysis as occurring in
molecule to an alcohol (Section 8.1.3). The
two stages:
fructose-6-phosphate is phosphorylated at the
Stage 1. This stage catalyzes the splitting of expense of ATP to fructose-1,6-bisphosphate
the glucose molecule (CG) into two phos- (FBP) by the enzyme fructose-6-phosphate
phoglyceraldehyde (C3) molecules. It con- kinase (reaction 3). The ATP used to phos-
sists of four consecutive reactions. Two phorylate fructose-6-phosphate is also regen-
ATPs are used per glucose metabolized; and erated from phosphoenolpyruvate in Stage
these donate the phosphoryl groups that 2. The fructose-1,6-bisphosphate is split into
become the phosphates in phosphoglyc- phosphoglyceraldehyde (PGALD) and dihy-
eraldehyde. (The phosphoglyceraldehyde droxyacetone phosphate (DHAP) by fructose-
eventually becomes phosphoenolpyruvate 1,6-bisphosphate aldolase (reaction 4). The
in stage two, and the phosphate that orig- splitting of fructose-1,6-bisphosphate is facil-
inated from ATP is returned to ATP in itated by the electron attracting keto group
the pyruvate kinase step, thus regenerating at C2, thus rationalizing the isomerization of
the ATP.) glucose-6-phosphate to fructose-6-phosphate
(Section 8.1.3). The dihydroxyacetone phos-
Stage 2. This stage catalyzes the oxidation of
phate is isomerized to phosphoglyceraldehyde
phosphoglyceraldehyde to pyruvate. It con-
(reaction 5), in a reaction similar to the earlier
sists of five consecutive reactions. Stage 2
isomerase reaction (reaction 2). Thus stage
generates four ATPs per glucose metabo-
lized, hence the net yield of ATP is two. 1 produces two moles of phosphoglyceralde-
hyde per mole of glucose.
Stage 2 reactions are not unique to glycoly-
In Stage 2, both moles of phosphogly-
sis and also occur when pyruvate is formed
ceraldehyde are oxidized to 1,3-bisphospho-
from phosphoglyceraldehyde in the pen-
glycerate (also called disphosphoglycerate,
tose phosphate pathway and the Entner-
DPGA) (reaction 6). The bisphosphoglycer-
Doudoroff pathway, accounting for ATP
synthesis in these pathways. ate serves as the phosphoryl donor for a
substrate-level phosphorylation catalyzed by
Stage 1: Glucose + 2ATP + 2PGALD + 2ADP the enzyme phosphoglycerate kinase (reac-
Stage 2: 2PGALD + 2Pj + 4ADP + 2NAD+ tion 7). At this point, two ATPs are made,
one from each of the two bisphosphoglyc-
+ 2 pyruvate + 4ATP + 2NADH + 2H+
erates. The product of the phosphoglycerate
182
H-C=O H-C=O
n
I {I 0 THzOH THzOC'V
H-y-OH ADP H-y-O-H y=O C=O
ADP I
HO-C-H AT.!:../ . ATP
I
H-C-OH 1
HO-C-H
I
H -C-OH
..
2
. HO-C- H
I
H-C-OH
'3 ./ · HO-C-H
(10
I I I H-y-O-H
H-C-OH H-C-OH H-C-OH
I I H-C-oH
I I
CHzOH CHZOC'V CHZOC'V CHzOC'V
glucose glucose-6-P fructose-6-P fructose-l,6-bisphosphate
H-C=O
I
o H-C-OH
II I
C-O-C'V CHzOC'V
I
H-C-OH 3-phosphoglyceraldehyde
I dihydroxy-
CHzo0
u,on,.,o,..."
1 ,3- bis phosphoglycerate
o
II
T-OH
H-C-OH
I
CHzOC'V
3-P-glycerate o
II o o
C-OH H20 II II
9 ~.
I C-OH A TP
r."
H -c-o-
L:'-' ~I
P
C'V I C1 ADP, ./.
C-OH
I
.. c-O-C'V 10 C=O
H-C- ro"H"
L:_..J
I e"CHz I
CH]
H
2-P-glycerate
phosphoenolpyruvate pyruvate
Fig. 8.2 Glycolysis. Enzymes: (1) hexokinase; (2) isomerase; (3) phosphofructokinase; (4) fructose-
1,6-bisphosphate aldolase; (5) triosephosphate isomerase; (6) triosephosphate dehydrogenase, (7)
phosphoglycerate kinase; (8) mutase; (9) enolase; (10) pyruvate kinase.
kinase reaction is 3-phosphoglycerate (3- glycolysis is coupled to the oxidation of phos-

PGA). The two moles of 3-phosphoglycerate phoglyceraldehyde to 3-phosphoglycerate (re-
are converted to two moles of 2-phosphogly- actions 6 and 7). For a more detailed discus-
cerate (2-PGA) (reaction 8), which are dehy- sion of substrate-level phosphorylations, see
drated to two moles of phosphoenolpyruvate Section 7.3.
(PEP) (reaction 9). The phosphoenolpyruvate
serves as the phosphoryl donor in a second site 8.1.1 Glycolysis as an
substrate-level phosphorylation to form two anabolic pathway
more moles of ATP and two moles of pyruvate
(reaction 10). Notice that the phosphate in The glycolytic pathway serves not only to
the 2-phosphoglycerate originated from ATP oxidize carbohydrate to pyruvate and to phos-
during the phosphorylations in stage 1 (re- phorylate ADP, but it also provides pre-
actions 1 and 3). Thus the net synthesis of cursor metabolites for many other path-
ATP from ADP and inorganic phosphate in ways. Figure 8.3 summarizes the glycolytic
183
reactions and points out only a few of the to fructose-1,6-bisphosphate, whereas the
branch points to other pathways. For ex- phosphatase catalyzes the dephosphoryla-
ample, glucose-6-phosphate is a precursor to tion of fructose-1,6-bisphosphate to fructose-
polysaccharides, pentose phosphates, and aro- 6-phosphate. Models for the regulation
matic amino acids; fructose-6-phosphate is of glycolysis are based primarily on in
a precursor to amino sugars (e.g., muramic vitro studies of the allosteric proper-
acid and glucosamine found in the cell wall), ties of the enzymes. Important effector
dihydroxyacetone phosphate is a precursor to molecules are AMP and ADP. When both
phospholipids, 3-phosphoglycerate is a pre- of these are high, ATP is low, since
cursor to the amino acids glycine, serine, and they are both derived from ATP. That is
cysteine; and phosphoenolpyruvate is a pre- to say,
cursor to aromatic amino acids and to the
lactyl portion of muramic acid. When the or- ATP ---4 ADP + Pi
ganisms are not growing on carbohydrate, ATP ---4 AMP+ PPi
they must synthesize these glycolytic interme-
diates from other carbon sources. Figure 8.3 Thus high ADP and AMP concentrations are
shows that some of the carbon from amino a signal that the ATP levels are low. (AI-
acids, carboxylic acids (organic acids), and losteric activation and inhibition are discussed
lipids is converted to phosphoenolpyruvate in Chapter 6.) Since glycolysis produces ATP,
from which the glycolytic intermediates can it makes sense to stimulate glycolysis when
be synthesized. Also, pyruvate can serve as the ATP levels are loW: E. coli accomplishes
a carbon source and therefore must be con- this by allosterically activating the phospho-
verted to glycolytic intermediates. However, fructokinase with ADP, which, as mentioned,
it can be seen that the glycolytic pathway is at a higher concentration when the ATP
can be reversed from phosphoenolpyruvate levels are low. At the same time that gly-
only to fructose-1,6-bisphosphate (FBP), and colysis is stimulated by ADP, the reversal
not at all from pyruvate. This is because of glycolysis is slowed by AMP, which is
the pyruvate kinase and phosphofructoki- also at a higher concentration when the ATP
nase reactions are physiologically irreversible levels are low. The reason for this is that
due to the high free energy in the phos- AMP inhibits the fructose-1,6-bisphosphate
phoryl donors with respect to the phospho- phosphatase reaction. The student may no-
rylated products. Therefore, to reverse gly- tice that the sum of the phosphofructoki-
co lysis the kinase reactions are bypassed. nase and fructose-1,6-bisphosphatase reaction
The conversion of fructose-1,6-bisphosphate is the hydrolysis of ATP (i.e., ATPase ac-
tivity). The stimulation of the phosphofruc-
to fructose-6-phosphate requires fructose-1,6-
bisphosphate phosphatase (Fig. 8.4). There tokinase by ADP and the inhibition of the
are also alternative ways to convert pyruvate the phosphatase by AMP prevents the un-
directly to phosphoenolpyruvate without us- necessary hydrolysis of ATP when the ATP
ing pyruvate kinase. These are discussed in levels are low. Glycolysis is not only reg-
Section 8.13. ulated by AMP and ADP in E. coli, but
also by phosphoenolpyruvate and fructose-
6-phosphate. As indicated in Fig. 8.3, the
8.1.2 Regulation of glycolysis phosphofructokinase is feedback inhibited by
phosphoenolpyruvate. This can be considered
Figure 8.3 also illustrates the regulation of an example of end-product inhibition. The
glycolysis in E. coli. Two key enzymes in pyruvate kinase, another physiologically ir-
regulating the directionality of carbon flow reversible reaction, is positively regulated by
are phosphofructokinase (reaction 1) and fructose-1,6-bisphosphate, which is an exam-
fructose-1,6-bisphosphate phosphatase (re- pie of a precursor metabolite activating a later
action 2), which catalyze physiologically ir- step in the pathway. (Feedback inhibition and
reversible steps. The kinase catalyzes the precursor activation are discussed in Sections
phosphorylation of fructose-6-phosphate 6.1.1 and 6.1.2.)
184
polysaccharides, pentose phosphates,

..
aromatic amino acids
.. amino sugars
I I
.. phospholipids
I I
.. serine, glycine, cysteine

I I
lipids, amino acids, .. PEP aromatic amino acids,

carboxylic acids muramic acid
0...PBP
3~
pyruvate
Fig. 8.3 Glycolysis as an anabolic pathway and its regulation in E. coli. The rationale for the pattern
of regulation is that when the ADP and AMP levels are high, the ATP levels are low and therefore
glycolysis is stimulated. Steady-state levels of intermediates are maintained by positive and negative
feedback inhibition.
8.1.3 The chemical bases for the it creates an electron attracting keto group at
isomerization and aldol cleavage C2 of the sugar, and the electron attracting
reactions in glycolysis keto group is necessary to break the bond
between C3 and C4 in the aldolase reaction.
It is important to understand the chemistry These reactions are shown in Fig. 8.5. The
of metabolic reactions as well as to learn isomerization can be viewed as the oxidation
the pathways and their physiological role. To of C2 by Cl, because two electrons shift
this end, the isomerization and aldol cleavage from C2 to Cl. This happens in two steps.
reactions will be explained because they are A hydrogen dissociates from C2 and two
common reactions that we will see later in electrons shift in to form the cis-enediol.
other pathways. Consider the isomerization of Then the hydrogen in the C2 hydroxyl
glucose-6-phosphate to fructose-6-phosphate. dissociates and two electrons shift in, forcing
The rationale for this isomerization is that the two electrons in the double bond to go
CHZoe!:) TH20H
I
C=O c=o
I I
HO-C-H HO-C-H
+ H20 · + P;
H-t-OH
I
H-t-OH
I
H-C-OH H-C-OH
I I
CHzoe!:) CHzOe!:)
fructose- ] ,6-bisphosphate fructose-6-phosphate
Fig. 8.4 The fructose-l,6-bisphosphatase reaction
185
to the Cl. The result is fructose-6-phosphate. to the acyl phosphate and the subsequent
The fructose-6-phosphate becomes phospho- transfer of the phosphoryl group to ADP). The
rylated to fructose-1,6-bisphosphate. The first reaction is catalyzed by triose-phosphate
fructose-1,6-bisphosphate is split by the al- dehydrogenase, and the second reaction is
dolase when the keto group on C2 pulls catalyzed by phosphoglycerate kinase. Given
electrons away from the C-C bond be- that these are the steps in which net ATP is
tween C3 and C4, as two electrons shift in made from ADP and inorganic phosphate,
from the hydroxyl on C4 (Fig. 8.5). The one can ask why the other intermediates
products of the split are phosphoglyceralde- are phosphorylated. Phosphorylation of the
hyde and dihydroxyacetone phosphate. A sec- intermediates requires the use of two ATPs
ond isomerization converts the dihydroxyace- in Stage 1, which are simply regenerated in
tone phosphate to phosphoglyceraldehyde via the pyruvate kinase step. There is probably
the same mechanism as the isomerization more than one reason why all of the
between glucose-6-phosphate and fructose- intermediates are phosphorylated. One reason
6-phosphate. In this way, two phosphoglyc- may be because the kinase reactions in
eraldehydes can be formed from glucose-6- Stage 1 are irreversible and therefore drive
phosphate. the reactions rapidly in the direction of
pyruvate. Another reason has to do with the
physiological role of the pathway. Glycolysis
8.1.4 Why are the glycolytic is not simply a pathway for the oxidation
intermediates phosphorylated? of glucose and the provision of ATP. Very
importantly, the glycolytic pathway also
In glycolysis, the phosphorylation of ADP by provides phosphorylated precursors to many
inorganic phosphate is due to two reactions other pathways. In subsequent chapters, we
that take place in Stage 2 (i.e., the oxidation of will study these interconnections with other
the C1 aldehyde of 3-phosphoglyceraldehyde pathways.
H-c:iO' H~-oH
H
I H
I
-
II ~ H-C-oH H-C-o(V
H'~-oH C~~H I I f\
¥ ,""- I I c=O C=O
HO-C-H
I
H-C-oH
I
H-C-oH
HO-C-H
H-C-oH
H-C-oH
I
I
- HO-C-H
I
I
H-C-oH
I
~ Hofi-H
H~~H
I
I I H-C-oH H-C-oH
CHp(V CHzO(V I I
glucose-6-P cis-enediol CHzO(V CHzO(V
fructose-6-P fructose- bisphosphate
~~~~
CHzO(V I
I H-C-oH
C~D I
II CHzO(V
(CHOH P-glyceraldehyde
CHZO(V
I
c=o
I
CHzOH
dihydroxyacetone-P
Fig. 8.5 Making two phosphoglyceraldehydes from glucose-6-phosphate. Glucose-6-phosphate itself
cannot be split because there is no electron attracting group to withdraw the electrons from the C-
C bond between carbons #3 and #4. An electron-withdrawing keto group is created on C2 when
glucose-6-phosphate is isomerized to fructose-6-phosphate.
186
8.2 The Fate of NADH is written NADH, not NADH2. To under-

stand why, we must examine the structures
If the NADH were not reoxidized to NAD+, (Fig. 8.6). Notice that the molecule can accept
then all pathways (including glycolysis) that two electrons but only one hydrogen. That is
require NAD+ would stop. Clearly, glycolysis why it is written as NADH + H+. The oxi-
must be coupled to pathways that re-oxidize dized molecule is written NAD+ because the
NADH back to NAD+. Bacteria have three nitrogen carries a formal positive charge.
ways to reoxidize NADH: respiration, fermen-
tation, and the hydrogenase reaction. Respira-
tion (aerobic or anaerobic):
8.4 A Modified EMP Pathway in
NADH + H+ + B + yADP + yP; the Hyperthermophilic Archaeon
---+ NAD+ + BH2 + yATP Pyrococcus furiosus
where y is approximately equal to the number
of coupling sites and B is the terminal electron The study of archaeal metabolism has recently
acceptor. (See Section 4.5.2 for a discussion been receiving wider attention, compared to
of the number of ATP molecules formed per the long history of research with bacteria,
coupling site according to the chemiosmotic and there have been many rewarding findings
theory. ) that suggest the presence of several metabolic
features distinct from those of the bacteria.3
Fermentation (anaerobic): Examples include the novel ether-linked lipids
described in Chapter 1 and their biosynthesis,
NADH + H+ + B (organic) ---+
discussed in Chapter 9. Additional features of
NAD+ + BH2 archaeal metabolism that appear to be unique
Hydrogenase (anaerobic): to these microorganisms include the synthesis
of methane, and the presence of novel coen-
zymes for acetate and methane metabolism
discussed in Chapter 13. A modified Embden-
Aerobic and anaerobic respiration are dis-
Meyerhof-Parnas pathway has been proposed
cussed in Chapter 4. In the absence of respira-
for Pyrococcus furiosus.4 This organism has a
tion, NADH can be reoxidized in the cytosol
growth temperature optimum of 100°C and
via fermentation discussed in Chapter 14. A
ferments carbohydrates and peptides to ac-
third way to reoxidize NADH is via the en-
etate, H2, and C02. Monosaccharides such as
zyme hydrogenase in the cytosol. Hydroge-
nases that use NADH as the electron donor
are found in fermenting bacteria. However, the Hi H
oxidation of NADH with the production of
~CONH2 ()-CONH2
hydrogen gas generally proceeds only when
the hydrogen gas concentration is kept low
tw.J N
;ibme ~ibos~
(e.g., during growth with hydrogen gas uti- I I
ADP ADP
lizers). This is because the equilibrium favors
NAD+ NADH
the reduction of NAD+. Interspecies hydrogen
transfer is discussed in Section 14.4.1. Fig. 8.6 The structures of NAD+, NADH,
and nicotinamide. NAD+ is a derivative of
nicotinamide, to which ADP-ribose is attached
8.3 Why Write NAD+ instead to the nitrogen of nicotinamide. In the oxidized
form the nitrogen has four bonds, and hence
ofNAD, and NADH instead carries a positive charge. NAD+ accepts two
ofNADH2? electrons, but only one hydrogen, (hydride
ion) to become NADH. The second hydrogen
Oxidized nicotinamide adenine dinucleotide removed from the electron donor (the reductant)
is written as NAD+, and the reduced form is released into the medium as a proton.
187
glucose and fructose do not support growth, glucose

but other carbohydrates such as maltose are ADP
hexokinaseV
transported into the cell and converted to (ADP)
glucose. If SO is present in the medium, it
f" AMP
glucose-6-phosphate
is used as an electron sink and reduced to
H2S. A pathway proposed for the catabolism 1
of glucose to acetate is shown in Fig. fructose-6-phosphate
8.7.5 The postulated pathway resembles the phospho- ADP
fructokinase
(ADP)
classic EMP pathway
respects. Very interestingly,
but differs in several
the phosphoryl
~ AMP
fructose-l,6-bisphosphate
donor in the hexokinase and fructokinase ~
reactions appears not to be ATP but rather glycernldehyde-3-phosphate
dihydroxyacetonephosphate
ADP, Another
that oxidizes
3-phosphoglycerate
difference
.
is that the enzyme
glyceraldehyde-3-phosphate
IS suggested to be a
to
GAPOR e-
~ Fd _n-'- FNOR _n-'- NADP
3-phO.'iphoglycerate
'- H -ase
~
211
~+ \H,
I
ferredoxin-linked enzyme rather than an
NAD+ -linked enzyme, and it appears that 1,3-
'
2- p hasp hog lycerate
..
blsphosphoglycerate
Pyruvate is oxidized
IS not
to
an intermediate.
acetyl-CoA and
enolase
t-
HzO
phosphoenolpyruvate
C02 by pyruvate-ferredoxin oxidoreductase. ADP

V
(See Section 7.3.2 for a description of this
f" ATP
reaction.) Finally, an ADP-dependent acetyl- pyruvate
CoA synthetase catalyzes a reaction in which f{~~~t:in
oxidoreductase
~
CoASH
ADP is phosphorylated and acetate is formed. ~-Fd _n-'- FNOR_n-'- NADpn_-,- H2-ase
See Section 7.3.2 for a description of the acetyl-CoA+C02 2H('\
H,
ADP-dependent acetyl-CoA synthetase and ADP+ P,
. .. . acetylCoA
ItS dlstnbUtion. All the enzymes proposed synth~tase
for the modified EMP pathway have been ~ ATP+CoASH
acetate
detected in extracts of P. furiosus, and in
vivo NMR studies of the products formed Fig. 8.7 Proposed pathway for maltose fer-
from [13CJglucose are in agreement with the mentation to acetate, C02, and H2 in Py-
pathway drawn in Fig. 8.7.5 rococcus furiosus. GAPOR, glyceraldehyde-
3-phosphate ferredoxin oxidoreductase; Fd,
ferredoxin; FNOR, ferredoxin NADP oxidore-
ductase; H2ase, hydrogenase. Source: Mukund,
8.5 The Pentose S., and M. W. W. Adams. 1995. Glyceraldehyde-
Phosphate Pathway 3-phosphate ferredoxin oxidoreductase, a novel
tungsten-containing enzyme with a potential gly-
Another important pathway for carbohydrate colytic role in the hyperthermophilic archaeon
metabolism is the pentose phosphate path- Pyrococcus furiosus. J. BioI. Chern. 270:8389-
. - 8392 .
way. Th e pentose p h asp h ate pat h way IS Im-
portant first because it produces the pentose
phosphates, which are the precursors to the
ribose and deoxyribose in the nucleic acids, NADPH.) The pathway is important to learn
and second because it provides erythrose phos- for yet one more reason. Several of the
phate, which is the precursor to the aro- reactions of the pentose phosphate pathway
matic amino acids, phenylalanine, tyrosine, are the same as the reactions in the Calvin
and tryptophan. Also, the NADPH produced cycle, which is used by many autotrophic
in the pentose phosphate pathway is a major organisms to incorporate C02 into organic
source of electrons for biosynthesis in most of carbon (Chapter 13).
the pathways in which reductions occur. (See The overall reaction of the pentose phos-
Note 6 for an alternative means of generating phate pathway is
188
G6P + 6 NADP+ > 3C02 + PGALD forms because the aldehyde group at C1 re-
+ 6 NADPH + 6 H+ (8.1) acts with the C5 hydroxyl group, forming a
hemiacetal. The C1 is therefore not a typ-
ical aldehyde in that it does not react in
8.5.1 The reactions of the pentose the Schiff test and does not form a bisul-
phosphate pathway fite addition product. Nevertheless, it is eas-
ily oxidized. The glucose-6-phosphate is ox-
The pentose phosphate pathway is complex idized by NADP+ to 6-P-gluconolactone by
and can be best learned by dividing the re- glucose-6-phosphate dehydrogenase (reaction
actions into three stages. Stage 1 consists 1). The lactone is then hydrolyzed to 6-P-
of oxidation-decarboxylation reactions. The gluconate by gluconolactonase (reaction 2).
C02 and NADPH are produced in Stage 1. In the oxidation of glucose-6-phosphate to 6-
Stage 2 consists of isomerization reactions that phosphogluconate, water contributes the sec-
make the precursors for Stage 3. Stage 3 reac- ond oxygen in the carboxyl group, and an
tions are sugar rearrangements. The phospho- acyl-phosphate intermediate is not formed.
glyceraldehyde is produced in Stage 3. This means that the energy from the oxida-
tion is lost as heat, and the reaction is phys-
iologically irreversible, as is often the case
Stage 1: Oxidation-decarboxylation
for the first reaction in a metabolic pathway.
reactions
(Recall that during the oxidation of phospho-
The oxidation-decarboxylation reactions are glyceraldehyde, inorganic phosphate is added
shown in Fig. 8.8. These reactions oxidize and 1,3-bisphosphoglycerate is formed. The
the C1 in glucose-6-phosphate to a carboxyl energy of oxidation is trapped in the acyl-
group and remove it as carbon dioxide. Glu- phosphate rather than being lost as heat.
cose actually exists as a ring structure, which A subsequent substrate-level phosphorylation
H-T
H-C-OH
HO-C-H
I
H-T-OH
H-C
~
I
OH
I
NAD(
NAoPH + W
1
)
·
H-~-OH
HO-C-H
I
H-T-OH
H-C
bl
-O
0
H-C-OH
HO-C-H
I
I
H-C-OH
I
COOH
I
I
I
H-C-OH
I I
CH200 CH200 CH200
glucose-6-P 6-P-gluconolactone 6-P-gluconate
NADP+
V
3~NADPH+W
CH20H COOH
I I
c=o H-C-OH
I I
H-C-OH c=o
I I
H-C-OH H-C-OH
I I
CH200 H-C-OH
I
ribulose-S-P (RuMP) CH200

3-keto-6-P-gluconate
Fig. 8.8 The oxidation-decarboxylation reactions. Enzymes: (1), glucose-6-phosphate dehydrogenase;

(2) gluconolactonase; (3) and (4) 6-phosphogluconate dehydrogenase.
189
recovers the energy of oxidation in the form to an aldose. The enzyme that catalyzes the
of ATP.) The product of the oxidation, 6- transfer of the two-carbon fragment is called
P-gluconate, is then oxidized on the C3 to a transketolase (TK). A second kind of reac-
generate a keto group fJ to the carboxyl tion transfers a three-carbon fragment from a
(reaction 3). A fJ-decarboxylation then occurs, ketose to an aldose. The enzyme that catalyzes
generating ribulose-S-phosphate (reaction 4). the transfer of a three-carbon fragment is
(The mechanism of fJ-decarboxylations is called a transaldolase (TA). The rule is that
described in Section 8.11.2.) Therefore, the the donor is always a ketose (with the OH
products of Stage 1 are carbon dioxide, 2 group of the third carbon "on the left," as
NADPH, and the five-carbon sugar phosphate in xylulose-S-phosphate) and the acceptor is
ribulose-S-phosphate. The rest of the pathway always an aldose. This rule is important to
continues with ribulose-S-phosphate. learn because we shall see other transketolase
and transaldolase reactions later. Knowing the
Stage 2: The isomerization reactions requirements will make it easier to remember
the reactions. The transketolase and transal-
During the second stage, some of the
dolase reactions are summarized in Fig. 8.10.
ribulose-S-phosphate is isomerized to ribose-
S -phosphate and to xylulose-S -phosphate.
Isomers are molecules having the same chem- Summarizing the pentose
ical formula but different structural formu- phosphate pathway
lae. That is to say, their parts have been
Figure 8.11 summarizes the pentose phos-
switched around. For example, the chem-
phate pathway. Reactions 1-3 comprise the
ical formula for ribulose-S-phosphate is
oxidative decarboxylation reactions of Stage
CSHll OsP. Ribose-S-phosphate and xylulose-
1. Three moles of glucose-6-phosphate must
S-phosphate have the same chemical for-
be oxidized in order to produce three moles
mula. However, their structures are different /
(Fig. 8.9). of C02 and one mole of phosphoglycer-

aldehyde. Therefore, Stage 1 produces three
One of the isomerases in an epimerase.
moles of ribulose-S-phosphate. Reactions 4
The epimerase catalyzes a movement of
and S are the isomerization reactions of Stage
the hydroxyl group from one side of the
2, in which the three moles of ribulose-S-
C3 in ribulose-S-phosphate to the other. The
phosphate are converted to one mole of ribose-
product is xylulose-S-phosphate (the epimer7
S-phosphate and two moles of xylulose-S-
of ribulose-S-phosphate). The other isomerase
phosphate. Reactions 6, 7, and 8 comprise
converts ribulose-S-phosphate to ribose-S-
Stage 3. Reaction 6 is a transketolase reac-
phosphate.
tion in which a xylulose-S-phosphate (Cs)
transfers a two-carbon moiety to ribose-
Stage 3: The sugar
S-phosphate (Cs) with the formation of
rearrangement reactions
sedoheptulose-7-phosphate (C7) and phos-
Stage 3 of the pentose phosphate pathway phoglyceraldehyde (C3). The two-carbon moi-
involves sugar rearrangement reactions. There ety is highlighted as a boxed area. Reaction
are two basic types of reactions. One kind 7 is a transaldolase reaction in which the
transfers a two-carbon fragment from a ketose sedoheptulose-7 -phosphate transfers a three-
yHzOH yHzOH H-C=O

I
c=o c=o H-C-OH
I I
I
HO-C-H
I
c . H-C-OH
I
.. 2 . H-C-OH
I
H-C-OH H-C-OH H-C-OH
I I I
CHzO(B CHzO(B CHzO(B
xylulose-S-P ribulose-S-P ribose-Sop
Fig. 8.9 The isomerization reactions. Enzymes: 1, RuMP epimerase; 2, ribose-5-phosphate isomerase.
190
~H20H
I -- I
C=O - -- - -- 'r:. H-C=O ;H20H

I ~ H-C=O 0( TK ..
I C=O
H-O--=e=-H
I H-C-OH + I I
\JI R2 I
I HO-C-H
H-C-OH
RI I
I R2
RJ
THPH
c=o __
- ''''1
TH20H
I - C=O
H-O-C-H H-C~O'
+ I .. TA.. + HO-C-H
,
H~e~O-H R2
I 'C./ H-C-OH
H-C-OH I
I R2
RI
Fig. 8.10 The transketolase and trans aldolase reactions. Enzymes: TK, transketolase; TA,
transaldolase. The donor is always a ketose with the keto group on C2, and the hydroxyl on C3
on the "left." In the transketolase reaction a C2 unit is transferred with its bonding electrons to the
carbonyl group on an aldehyde acceptor. The transaldolase transfers a three-carbon fragment. In the
transketolase reaction, the newly formed alcohol group is on the "left," which means that the products
of both the transketolase and transaldolase reactions can act as donors in a subsequent transfer.
carbon moiety to the phosphoglyceralde- The result is that three moles of glucose-
hyde to form erythroseA-phosphate (C4) and 6-phosphate are converted to two moles
fructose-6-phosphate (C6). The three-carbon of fructose-6-phosphate and one mole of
moiety is highlighted as a dashed box. Re- phosphoglyceraldehyde. The two moles
action 8 is a trans keto lase reaction in which of fructose-6-phosphate become glucose-6-
xylulose-5-phosphate transfers a two-carbon phosphate by isomerization, and the net result
moiety to the erythroseA-phosphate, form- is the conversion of one mole of glucose-6-
ing phosphoglyceraldehyde and fructose-6- phosphate to one mole of phosphoglyceralde-
phosphate. You will notice that the sequence hyde, three moles of carbon dioxide, and six
of reactions is: moles ofNADPH. This is shown in the carbon
balance below.
Transketolase ---+ transaldolase The carbon balance for the pentose phos-
---+ transketolase phate pathway is:
Oxidative decarboxylation 3 glucose-6-P + 3 ribulose-S-P + 3C02

3C6 3C5 3C1
Isomerizations 3 ribulose-S-P + 2 xylulose-S-P + ribose-S-P

3C, lCi C\
Transketolase xylulose-S-P + ribose-S-P + sedoheptulose-7-P + phosphoglyceraldehyde

C, C, C7 C,
Transaldolase sedoheptulose-7-P + phosphoglyceraldehyde + fructose-6-P + erythrose-4-P

C7 C3 C6 C4
Transketolase xylulose-S-P + erythrose-4-P + fructose-6-P + phosphoglyceraldehyde

~ ~ ~ G
Sum: glucose-6-P + phosphoglyceraldehyde + 3C02

C6 C, 3C1
191
Some bacteria rely comp!etely on the (e.g., gluconic acid), they use the Entner-
pentose phosphate pathway for Doudoroff pathway. However, some strictly
sugar catabolism aerobic bacteria cannot carry out Stage 1
of the Embden-Meyerhof-Parnas pathway
Thiobacillus novellus and Brucella abortus
lack both Stage 1 of the Embden-Meyerhof- and rely entirely on the Entner-Doudoroff
Parnas pathway and the enzymes of the pathway for sugar degradation (Table 8.1).
Entner-Doudoroff pathway. These organisms The overall reaction for the Entner-Doudoroff
use only an oxidative pentose phosphate path- pathway is
way to grow on glucose. They oxidize the glu-
glucose + NADP+ + NAD+ + ADP + Pi
coseto phosphoglyceraldehyde via the pentose
phosphate pathway. The phosphoglyceralde- ~ 2 pyruvic acid + NADPH + 2H+
hyde is then oxidized to pyruvate via reactions + NADH + ATP
that are the same as Stage 2 of the EMP path-
It can be seen that the pathway catalyzes
way, and then the pyruvate is oxidized to C02
the same overall reaction as the Embden-
via the citric acid cycle.
Meyerhoff-Parnas pathway, (i.e., the oxida-
tion of one mole of glucose to two moles
Relationship of the pentose phosphate
pathway to glycolysis of pyruvic acid), except that only one ATP
is made, and one NADPH and one NADH
The pentose phosphate pathway and glycol- are made instead of two NADHs. The reason
ysis interconnect at phosphoglyceraldehyde why only one ATP is made is that only one
and fructose-6-phosphate (Fig. 8.1). Thus or- phosphoglyceraldehyde is made from glucose
ganisms growing on pentoses can make hex- (Fig. 8.12).
ose phosphates. Furthermore, because Stages
2 and 3 of the pentose phosphate pathway are
reversible, it is possible to synthesize pentose 8.6.1 The Entner-Doudoroff reactions
phosphates from phosphoglyceraldehyde and
avoid the oxidative decarboxylation reactions The first oxidation is the oxidation of the Cl
of Stage 1. This would uncouple pentose phos- in glucose-6-phosphate to the carboxyl in 6-P-
phate synthesis from NADPH production, and gluconate (Fig. 8.12, reaction 2). These are the
confer a possibly advantageous metabolic flex- same enzymatic reactions that oxidize glucose-
ibility on the cells. 6-phosphate in the pentose phosphate path-
way, and proceed through the gluconolactone.
8.6 The Entner-Doudoroff

Table 8.1 Distribution of the
Pathway Embden-MeyerhoH-Parnas (EMP) and
Entner-doudoroH (ED) pathways in
Many prokaryotes have another pathway certain bacteria.
for the degradation of carbohydrates called
the Entner-Doudoroff or ED pathway. The Bacterium EMP ED
other pathways that we have been studying
Arthrobacter species +
are common to all cells, whether they be
Azotobacter chroococcum +
prokaryotes or eukaryotes, but the Entner- Alcaligenes eutrophus +
Doudoroff pathway has been found almost Bacillus species +
entirely among the prokaryotes, including Escherichia coli and other
bacteria and archaea (reviewed in Ref. 1). The enteric bacteria a +
Pseudomonas species +
pathway is widespread, particularly among
Rhizobium species +
the aerobic gram-negative bacteria. It is Thiobacillus species +
usually not found among anaerobic bacteria, Xanthomonas species +
perhaps because of the low ATP yields
a Organisms such as E. coli synthesize the enzymes of
discussed below. Most bacteria degrade sugars
the ED pathway when growing on gluconate.
via the Embden-Meyerhof-Parnas pathway, Source: Gottschalk, G. 1986. Bacterial Metabolism.
but when grown on certain compounds Springer-Verlag, New York, Berlin.
193
H-C=O H-C=O
o
I I II
H-C-OH H-C-OH C-OH
I ADP NADPH + H+ I
I
)
OH-C-H
I
H-C-OH
Al(
1 · OH-C-H
I
H-C-OH
NAD~
~2 HP,
H-C-OH
OH-C-H
I
I
I I
H-C-OH H-C-OH
I H-C-OH I
I H-C-OH
CH20H CH200 I
glucose glucose-6-P CHP0
6-P-gluconate
o
II
C-OH
I
C=O
I
CHJ
o
II
H-C=O
I '\,.,.:' o
II
C-OH H-C-OH C-OH
I I 'I
C=O CH20(E) .... C=O
I I
P-glyceraldehyde CH2
CHJ
NAD+ I
pyruvate
NADH + H+ H-C-OH
ATP-
AD~9
t P
".1
5 H-C-OH
I
I
CHp(E)
2-keto-3-deoxY-6_P.
o gluconate [KDPG]
II o
C-OH II
I C-o-(E)
C-o-(E) I
II H-C-OH
CH2 I
CHp(E)
phosphoenol-
H20 pyruvate 1,3-bisphosphoglycerate
ADP
8 6
~o
ATP
~ o
II II
C-OH C-OH
I 7 I
H-C-O-(E) H-C-OH
I I
CH20H CH20(E)
2-P-glycerate 3-P-glycerate
Fig. 8.12 The Entner-Doudoroff pathway. Because there is only one PGALD formed, there is only one
ATP made. The enzymes unique to this pathway are the 6-phosphogluconate dehydratase (reaction
3) and the KDPG aldolase (reaction 4). The other enzymes are present in the pentose phosphate
pathway and the glycolytic pathway. Enzymes: 1, hexokinase; 2, glucose-6-phosphate dehydrogenase;
3, 6-phosphogluconate dehydratase; 4, KDPG aldolase; 5, triose phosphate dehydrogenase; 6, PGA
kinase; 7, mutase; 8, enolase; 9, pyruvate kinase.
The pathway diverges from the pentose phos-

KDPG. This is illustrated in Fig. 8.13. In this
phate pathway at this point because some of way, it is similar to the dehydration of 2-
the 6-P-gluconate is dehydrated to 2-keto- phosphoglycerate to phosphoenolpyruvate by
3-deoxy-6-P-gluconate (KDPG), rather than enolase described in Section 7.3.4. However,
being oxidized to ribulose-5-phosphate (reac- in phosphoenolpyruvate, the enol derivative
tion 3). The KDPG is split by KDPG aldolase is stabilized by the phosphate group, and the
to pyruvate and phosphoglyceraldehyde (re- tautomerization does not take place until the
action 4). The phosphoglyceraldehyde is ox- phosphoryl group is transferred.
idized to pyruvate in a sequence of reactions
identical to those in Stage 2 of the EMP path-
way (reactions 5-9). 8.6.2 Physiological role for the
Reaction 3, which is the dehydration of
Entner-Doudoroff pathway
6-phosphogluconate to KDPG, takes place
Since the Entner-Doudoroff pathway pro-
via an enol intermediate that tautomerizes to
duces only one ATP, one can ask why the
194
COO COO. COO.

I I
H-C-OH
I
HO-C-H
~-OH
C-H
. C=O
I
TH2
I I
R
r R j R
Fig. 8.13 Dehydration of a carboxylic acid with hydroxyl groups in the ex and {3 position. The
dehydration of a carboxylic acid with hydroxyl groups in both the ex and {3 position leads to the
formation of an enol, which tautomerizes to the keto compound. That is because the hydroxyl on
the C3 leaves with its bonding electrons and the electrons bonded to the hydrogen on the C2 shift in
to form the double bond. This happens when 6-phosphogluconate is dehydrated to 2-keto-3-deoxy-
6-phosphogluconate in the Entner-Doudoroff pathway, and when 2-phosphoglycerate is dehydrated
to phosphoenolpyruvate during glycolysis. The phosphoenolpyruvate tautomerizes to pyruvate when
the phosphate is removed during the kinase reaction.
pathway is so common in the bacteria. gluconate may confer a competitive ad-

Whereas hexoses are readily degraded by the vantage, since it removes glucose, which
Embden-Meyerhoff-Parnas pathway, aldonic is more readily utilizable by other micro-
acids (aldoses oxidized at the aldehydic orgamsms.
carbon) such as gluconate are not, but
can be degraded via the Entner-Doudoroff
8.6.3 A partly nonphosphorylated
pathway. (Aldonic acids occur in nature and
Entner-Doudoroff pathway
can be an important nutrient.) An example
of this occurs when E. coli is transferred
A modified ED pathway has been found
from a medium containing glucose as the
in the archaeon Halobacterium saccharovo-
carbon source to one in which gluconate is
rum, and in several bacteria, including mem-
the source of carbon. Growth on gluconate
bers of the genera Clostridium, Alcaligenes,
results in the induction of three enzymes: a
Achromobacter, and in Rhodopseudomonas
gluconokinase that makes 6-P-gluconate from
sphaeroides. The pathway is characterized
the gluconate (at the expense of ATP), 6-P-
as having nonphosphorylated intermediates
gluconate dehydratase, and KDPG aldolase.
prior to 2-keto-3-deoxygluconate. The first
Thus E. coli uses the Entner-Doudoroff
reaction is the oxidation of glucose to glu-
pathway to grow on gluconate and the
conate via an NAD+ -dependent dehydroge-
Embden-Meyerhoff-Parnas pathway to grow
nase. The gluconate is then dehydrated to
on glucose.
2-keto-3-deoxygluconate by a gluconate dehy-
dratase. The 2-keto-3-deoxygluconate is phos-
Some bacteria do not have a complete phorylated by a special kinase to form KDPG,
Embden-Meyerhoff-Parnas pathway which is metabolized to pyruvate using the
and rely on the Entner-Doudoroff ordinary ED reactions. Those bacteria that
pathway for hexose degradation
have a modified ED pathway can use it for
Several prokaryotes (e.g., the pseudomon- the catabolism of gluconate, or glucose if the
ads in general) do not make phospho- glucose dehydrogenase is present.
fructokinase. Hence they cannot carry
out glucose oxidation using the Embden-
Meyerhoff-Parnas pathway (EMP). Instead, 8.7 The Oxidation of Pyruvate to
they use the Entner-Doudoroff pathway acetyl-CoA: The Pyruvate
(ED). See Table 8.1 for the distribution Dehydrogenase Reaction
of the EMP and ED pathways. Some of
the pseudomonads even oxidize glucose Pyruvate is the common product of sugar
to gluconate before degrading it via the catabolism in all the major carbohydrate
ED pathway, instead of making glucose- catabolic pathways (Fig. 8.1). We now exam-
6-phosphate. The oxidation of glucose to ine the metabolic fate of pyruvate. One of the
195
fates of pyruvate is to be oxidized to acetyl- The individual reactions carried out by the
CoA. As described in Section 7.3.2, this is pyruvate dehydrogenase complex are as fol-
catalyzed in anaerobically growing bacteria lows (Fig. 8.14):
by either pyruvate-ferredoxin oxidoreductase Step 1. Pyruvate is decarboxylated to form
or pyruvate-formate lyase. The acetyl-CoA "active acetaldehyde" bound to TPP (Fig.
thus formed is converted to acetyl-phosphate 8.14). The reaction is catalyzed by pyruvate
via the enzyme phosphotransacetylase, and dehydrogenase (E1). (The mechanism of
the acetyl-phosphate donates the phosphoryl this reaction is described in Section 14.10.)
group to ADP in a substrate-level phosphory-
Step 2. The "active acetaldehyde" is oxidized
lation catalyzed by acetate kinase. The acetate to the level of carboxyl by the disulfide
thus formed is excreted. (However, certain in lipoic acid. The disulfide of the lipoic
anaerobic archae a oxidize pyruvate to acetyl- acid is reduced to a sulfhydryl. During the
CoA using pyruvate-ferredoxin oxidoreduc- reaction, TPP is displaced and the acetyl
tase and oxidize the acetyl-CoA to C02 via group is transferred to the lipoic acid.
the citric acid cycle. 10)The oxidation of pyru- The reaction is also catalyzed by pyruvate
vate to acetyl-CoA during aerobic growth is dehydrogenase.
carried out by the enzyme complex pyruvate
dehydrogenase, which is widespread in both Step 3. A transacetylation occurs where lipoic
prokaryotes and eukaryotes. (The acetyl-CoA acid is displaced by CoASH, forming
acetyl-CoA and reduced lipoic acid. The
formed during aerobic growth is oxidized to
reaction is catalyzed by dihydrolipoate
C02 in the citric acid cycle.) The overall reac-
transacetylase, E2.
tion for pyruvate dehydrogenase is:
Step 4. The lipoic acid is oxidized by
CHjCOCOOH + NAD+ + CoASH dihydrolipoate dehydrogenase, E3-FAD.
+ CH3COSCoA+ C02 + NADH+ H+ Step 5. The E3-FADH2 transfers the electrons
The pyruvate dehydrogenase complex is a very to NAD+.
large enzyme complex (in E. coli about 1.7 All the intermediates remain bound to the
times the size of the ribosome) located in the complex and are passed from one active site
mitochondria of eukaryotic cells and in the to another. Presumably, this has the advantage
cytosol of prokaryotes. The pyruvate dehydro- inherent in all multienzyme complexes (i.e.,
genase from E. coli consists of 24 molecules there is no dilution of intermediates in the
of enzyme El (pyruvate dehydrogenase), cytosol, and side reactions are minimized).
24 molecules of enzyme E2 (dihydrolipoate The student should refer to Section 1.2.6 for
transacetylase), and 12 molecules of enzyme a discussion of multienzyme complexes in
E3 (dihydrolipoate dehydrogenase). Several the cytoplasm.
very important cofactors are involved. The
cofactors are thiamine pyrophosphate (TPP)
derived from the vitamin thiamine, flavin ade-
8.7.1 Physiological control
nine dinucleotide (FAD) derived from the vita- The pyruvate dehydrogenase reaction, which
min riboflavin, lipoic acid (RS2), nicotinamide is physiologically irreversible, is under
adenine dinucleotide (NAD+) derived from metabolic control by several allosteric effec-
the vitamin nicotinamide, and coenzyme-A, tors (Fig. 8.15). The E. coli pyruvate de-
derived from the vitamin pantothenic acid. hydrogenase is feedback inhibited by the
(See Note 8 for a more complete discussion products it forms, acetyl-CoA and NADH.
of vitamins.) The large size of the complex This can be rationalized as ensuring that the
is presumably designed to process the heavy enzyme produces only as much acetyl-CoA
stream of pyruvate that is generated during the and NADH as can be used immediately. It is
catabolism of sugars and other compounds. also stimulated by phosphoenolpyruvate (the
As described below, the pyruvate dehydro- precursor to pyruvate), presumably signaling
genase complex catalyzes a short metabolic the dehydrogenase that more pyruvate is on
pathway rather than simply a single reaction. the way. It is also stimulated by AMP, which
196
It by the
re as fol- EI-TPP +
to form
[PP (Fig. OH
I
pyruvate El-TPP-<r-CH3
mism of H
"active acetaldehyde"
n 14.10.)
oxidized
disulfide E2-R~ 2
he lipoic
lIring the
I
Redox I
a
II
le acetyl E2_R~-C-CHJ
)ic acid. 'sH
pyruvate CoASH
E2-R.»H
~relipoic 'sH
forming
cid. The
'olipoate
ized by
-FAD.
~Iectrons Fig. 8.14 The pyruvate dehydrogenase complex (PDH). Step 1. Pyruvate is decarboxylated to "active
acetaldehyde." The decarboxylation requires thiamine pyrophosphate (TPP). Step 2. The "active
:l to the acetaldehyde" is oxidized to an acylthioester with a high acyl group transfer potential. The oxidant is
reduced lipoic acid (R-S2). Step 3. The lipoylacylthioester transfers the acetyl group to coenzyme
:tive site
A (CoASH) to form acetyl-CoA. Step 4. The reduced lipoic acid is reoxidized by FAD, which
Jvantage
in turn is reoxidized by NAD+ (Step 5). The products of the reaction are acetyl-CoA, C02, and
xes (i.e., NADH. Enzymes: EI, pyruvate dehydrogenase; E2, dihydrolipoate transacetyase; E3, dihydrolipoate
s in the dehydrogenase.
limized).
1.2.6 for signals low ATP. The stimulation by AMP Notice that there are four oxidations per
lexes in probably reflects the fact that the oxidation acetyl-CoA producing two NADH, one
of the product acetyl-CoA in the citric acid NADPH, and one FADH2, and one substrate-
cycle is a major source of ATP (via respiratory level phosphorylation producing ATP. The
phosphorylation). cycle usually operates in conjunction with
respiration that reoxidizes the NAD(P)H and
1, which
FADH2. Other names for this pathway are the
under
tricarboxylic acid (TCA) cycle and the Krebs
ic effec-
8.8 The citric acid cycle cycle. The latter name honors Sir Hans Krebs,
vate de-
by the who did much of the pioneering work and
The acetyl-CoA that is formed by pyruvate proposed the cycle in 1937.
NADH. dehydrogenase is oxidized to C02 in the citric
that the acid cycle (Fig. 8.1). The overall reaction is:
tyl-CoA
ely. It is acetyl-CoA + 2H20 + ADP + Pi + FAD 8.8.1 The individual reactions of the
rate (the citric acid cycle
+ NADP+ + 2NAD+
ignaling
te is on
+ 2C02 + ATP + FADH2+ NADPH The pathway is outlined in Fig. 8.16. Reaction
J, which + 2NADH + 3H+ + CoASH 1 is the addition of the acetyl group from
197
'f decarboxylation of pyruvate to acetyl-CoA.

PEP
Reaction 7 (succinate thiokinase) is a
.
pyruvate substrate-level phosphorylation resulting in
NAD 0 NADH
the formation of ATP from ADP and inorganic
0 acetyl-CoA phosphate. This reaction was described in Sec-
NADH
CoASH
q
acetyl-CoA
0
0
+ C02
PEP
AMP
tion 7.3.3. Reaction 8 (succinate dehydroge-
nase) is the oxidation of succinate to fumarate,
catalyzed by a flavin enzyme. Succinate dehy-
drogenase is the only citric acid cycle enzyme
Fig. 8.15 Regulation of pyruvate dehydrogenase
that is membrane bound. In bacteria it is part
in E. coli. The activity of the enzyme in vitro is
of the cell membrane and transfers electrons
modified by several effector molecules. NADH
and acetyl-CoA are negative effectors, and PEP directly to quinone in the respiratory chain.
and AMP are positive effectors. (See the description of the electron transport
chain in Section 4.3.) Reaction 9 (fumarase)
is the hydration of fumarate to malate. Fi-
acetyl-CoA to oxaloacetate to form citrate. In
nally, the oxaloacetate is regenerated by oxi-
this reaction the methyl group of acetyl-CoA
dizing the malate to oxaloacetate in reaction
acts as a nucleophile and bonds to the carbon
10 (malate dehydrogenase). The citric acid
in the keto group of oxaloacetate (OAA). The
cycle proceeds in the direction of acetyl-CoA
reaction is driven to completion by the hy-
oxidation because of two irreversible steps:
drolysis of the thioester bond of acetyl-CoA,
the citrate synthase and the a-ketoglutarate
which has a high free energy of hydrolysis. Re- dehydrogenase reactions.
action 1 is catalyzed by citrate synthase. This
enzyme operates irreversibly in the direction
Summing up the citric acid cycle
of citrate. The oxaloacetate acts catalytically
in the cycle, and, if it is not regenerated or When one examines the reactions in the citric
replenished, the pathway stops. Examine the acid cycle, it can be seen that there is no
structure of citrate shown in Fig. 8.16. In the net synthesis. In other words, all the carbon
subsequent reactions of the citric acid cycle, that enters the cycle exits as C02. This is
the C3 and CS carbons will be removed as C02 made clear by writing a carbon balance. In the
and the C1-CS carbons will regenerate the ox- carbon balance written below, C2 represents
aloacetate. In reaction 2, catalyzed by aconi- the two-carbon molecule acetyl-CoA, C6
tase, the citrate is dehydrated to cis-aconitate, represents citrate or isocitrate, Cs represents
which remains bound to the enzyme. Reac- a-ketoglutarate, and C4 represents either
tion 3 (also catalyzed by aconitase) is the re- succinate fumarate, malate, or oxaloacetate.
hydration of cis-aconitate to form isocitrate, Of course, C1 represents carbon dioxide.
an isomer of citrate. In reaction 4 (isocitrate Carbon balance for citric acid cycle:
dehydrogenase) the isocitrate is oxidized to
oxalosuccinate. This oxidation creates a keto C2 + C4 + C6
F
group fJ to the carboxyl group. The creation C6 + Cs + C1
d
of the keto group is necessary for the decar- Co + C4 + C1 d
boxylation that takes place in the next reac- b
sum: C2
tion (fJ-keto decarboxylations are explained in
Section 8.11.2).
Reaction 5 (isocitrate dehydrogenase) is 8.8.2 Regulation of the citric
the decarboxylation of oxalosuccinate to a- acid cycle
ketoglutarate. Reaction 6 (a-ketoglutarate r
dehydrogenase) is the oxidative decarboxy- The citric acid cycle is feedback inhibited
lation of a-ketoglutarate to succinyl-CoA. by several intermediates that can be viewed
This is an a-decarboxylation, in contrast to as end products of the pathway. In gram- a
a fJ-decarboxylation. It is a complex reaction negative bacteria, the citrate synthase is t
and requires the same cofactors as does the allosterically inhibited by NADH, and in a
198
3COOH
4t=0
5tH 2
I
6COOH
INADH + oxaloacetate
I
WI",I
NAD+110
COOH COOH
I I
OH-C-H 'rH2
I
C-COOH
'rH2 II
COOH H-C
L-malate I
COOH
cis-aconitate
~ " f.9 H20
3/
COOH \
~H
II
'r~
H-C CH2
I
I H-C-COOH
COOH I
fumarate
HO-C-H
~FADH2 8
tOOH
isocitrate
~
FAD
'rOOH
4
~
NADP+
CH2 COOH
r INADPH+wl
I
I
'rH2 yH2
COOH SUCcinate
H-'r-COOH
~
c=o
CoASH 7 tOOH
'rOOH oxalosuccinate
+~ C~
I
C-SCoA
I
CH2
I
;<I CO
2 I
II CH2
~
o
succinyl-CoA 6 t=O
I
COOH
~CO2 NAD+ '\ a-ketoglutarate
CoASH
NADH + H+
I I
Fig. 8.16 The citric acid cycle. Enzymes: (1) citrate synthase; (2, 3) aconitase; (4, 5) isocitrate
dehydrogenase; (6), a-ketoglutarate dehydrogenase; (7), succinate thiokinase; (8), succinate
dehydrogenase; (9), fumarase; (10), malate dehydrogenase. cis-Aconitate is drawn in parentheses
because it is an enzyme-bound intermediate.
facultative anaerobes such as E. coli, also a-ketoglutarate.9 The citrate synthase from
by a-ketoglutarate. The inhibition of the cit- gram-positive bacteria and eukaryotes is not
rate synthase by NADH may be a way to sensitive to NADH and a-ketoglutarate but
prevent oversynthesis of NADH. The inhibi- is inhibited by ATP, another end product of
tion by a-ketoglutarate can also be viewed the citric acid pathway. Recall the discussion
as an example of end-product inhibition, in in Chapter 6 emphasizing that the pattern of
this case to prevent overproduction of the regulation of a particular pathway need not be
amino acid glutamate, which is derived from the same in different bacteria.
199
8.8.3 The citric acid cycle as an not necessary for these organisms since or the ot
anabolic pathway acetyl-CoA is not an intermediate in the animal ti
oxidation of the C1 compounds. Although
The citric acid cycle reactions provide pre- the oxidative citric acid cycle is a pathway
cursors to 10 of the 20 amino acids found usually associated with aerobic bacteria, it
8.9.1 R4
in proteins. It is therefore a multifunctional is also present in certain anaerobes that
pathway and is not used simply for the ox- In E. colI
oxidize organic compounds completely to
zyme the
idation of acetyl-CoA. Succinyl-CoA is nec- C02. These include the group II sulfate
CoA all(
essary for the synthesis of the amino acids L- reducers (discussed in Section 12.2.2) and
(Fig. 8.1
lysine and L-methionine. Succinyl-CoA is also certain archaea. The latter are anaerobic
levels dn
a precursor to tetrapyrroles, which are the hyperthermophilic archaea that use sulfur or
and resul
prosthetic group in several proteins, including thiosulfate as the terminal electron acceptor
lase. Thi:
cytochromes and chlorophylls. Oxaloacetate (Ref. 10).
Aspartatl
is a precursor to the amino acid aspartate,
negativel
which itself is the precursor to five other amino
acids. In some bacteria, fumarate is also a 8.9 Carboxylations that Replenish
precursor to aspartate. a-Ketoglutarate is the
Oxaloacetate: The Pyruvate and 8.l0M
precursor to the amino acid glutamate, which
itself is the precursor to three other amino
Phosphoenolpyruvate Carboxylases Cyclei
acids. The biosynthesis of amino acids is de-
Because the citric acid cycle intermediates are
Cyclec
scribed in Chapter 9. However, since the citric
constantly being removed to provide precur-
acid cycle requires a constant level of oxaloac- In the pfl
sors for biosynthesis, they must be replaced
etate in order to function, net synthesis of ates as al
(Section 8.8.3). Failure to do this would de-
these molecules requires replenishment of the bic respil
crease the level of oxaloacetate that is nec-
oxaloacetate. This is discussed in Section 8.9. fermenti
essary for the citrate synthase reaction, and
obic res]
thus for the continuation of the cycle. If the
8.8.4 Distribution of the citric organism is growing on amino acids or or- an oxida
NADH.
acid cycle ganic acids (e.g., malate), then replenishment
reactiom
of oxaloacetate is not a problem, since these
CoA, an
The citric acid cycle is present in most molecules are easily converted to oxaloacetate
heterotrophic bacteria growing aerobically. (Fig. 8.27). If the carbon source is a sugar (e.g., cause tb
However, not all aerobic bacteria have a com- glucose), then the carboxylation of pyruvate
plete citric acid cycle. For example, organ- or phosphoenolpyruvate replenishes the ox-
isms that grow on C1 compounds (methane, aloacetate (Fig. 8.17). Two enzymes that carry
methanol, and so on described in Chapter out the carboxylation of phosphoenolpyru-
13) lack a-ketoglutarate dehydrogenase and vate and pyruvate are PEP carboxylase and
carry out a reductive pathway as described in pyruvate carboxylase, which are widespread
Section 8.9. An oxidative citric acid cycle is among the bacteria. A bacterium will have one
COOH
COOH I
I C=O
~=o + ATP + H*C03- . I + ADP + P,
~H2
CH3
pyruvate *COOH
oxaloacetate
COOH
COOH I
I
c-o-0
II
+ H*CO 3-
2 .. C=O
I + P,
CH2 ~H2
phosphoenolpyruvate *COOH
oxaloacetate
Fig. 8.U
.
E. coli.
Fig. 8.17 Carboxylation reactions that replenish the supply of oxaloacetate. Enzymes: 1, pyruvate by acet:
carboxylase; 2, PEP carboxylase. Bacteria may have one or the other. aspartatl
200
;alllsms SInce or the other. PEP carboxylase is not found in biosynthesis of amino acids and tetrapyrroles.
ediate in the animal tissues or in fungi. (See Section 8.8.3 and Fig. 8.27.) The solu-
lds. Although tion to the problem is to convert the citric
is a pathway acid cycle from an oxidative into a reductive
8.9.1 Regulation of PEP carboxylase
ic bacteria, it pathway. The reductive pathway is also re-
laerobes that ferred to as an incomplete citric acid cycle.
In f. coli PEP carboxylase is an allosteric en-
:ompletely to Fermenting bacteria have little or no activ-
zyme that is positively regulated by acetyl-
up II sulfate ity for the enzyme a-ketoglutarate dehydro-
CoA and negatively regulated by aspartate
I 12.2.2) and genase. Thus the pathway is blocked between
(Fig. 8.18). Presumably, if the oxaloacetate
Ire anaerobic a-ketoglutarate and succinyl-CoA, and can-
levels drop, then acetyl-CoA will accumulate
: use sulfur or not operate in the oxidative direction (Fig.
and result in the activation of the PEP carboxy-
:tron acceptor 8.19). Succinyl-CoA is made by reversing the
lase. This should produce more oxaloacetate.
reactions between oxaloacetate and succinyl-
Aspartate can slow down its own synthesis by
CoA, using the enzyme fumarate reductase
negatively regulating PEP carboxylase.
instead of succinate dehydrogenase. The lat-
Replenish ter enzyme is replaced by fumarate reductase
late and under anaerobic conditions. These reactions
8.10 Modification of the Citric Acid consume 4H. If one includes the reactions
lrboxylases Cycle into a Reductive (Incomplete) from citrate to a-ketoglutarate that produce
ermediates are
Cycle during Fermentative Growth 2H, the net result is that the reductive pathway
consumes 2H. The reductive citric acid path-
rovide precur-
In the presence of air, the citric acid cycle oper- way is found not only in fermenting bacteria,
st be replaced
ates as an oxidative pathway coupled to aero- but also in some other bacteria (including the
his would de-
bic respiration in respiratory organisms. Since enteric bacteria) that are carrying out anaer-
:e that is nec-
fermenting organisms are not carrying out aerobic respiration using nitrate as the electron
reaction, and
obic respiration, it seems best not to have acceptor. II The reason for this is that oxygen
Ie cycle. If the
an oxidative pathway that produces so much induces the synthesis of a-ketoglutarate de-
o acids or or-
NADH and FADH2. On the other hand, the hydrogenase in certain facultative anaerobes,
replenishment
reactions that make oxaloacetate, succinyl- and under anaerobic conditions, the enzyme
:ill, since these
CoA, and a-ketoglutarate are necessary be- levels are very low. (However, some nitrate
o oxaloacetate
cause these molecules are required for the respirers do have an oxidative citric acid cy-
is a sugar (e.g.,
cle (e.g., Pseudomonas stutzeri grown under
m of pyruvate
denitrifying conditions.12)
nishes the ox-
One of the consequences of an incomplete
rmes that carry
citric acid cycle is that acetate is excreted
sphoenolpyru-
as a by-product of sugar metabolism during
rboxylase and
anaerobic growth. That is because some
Ire widespread
of the acetyl-CoA is converted to acetate
n will have one
concomitant with the formation of an ATP.
These reactions, which are an important
source of ATP, are discussed in Chapter
14. It should be pointed out that some
strict anaerobes (e.g., the green photosynthetic
sulfur bacteria) have a reductive citric acid
pathway that is "complete" in that it reduces
oxaloacetate to citrate. The pathway, called
the reductive carboxylic acid pathway, is a
C02 fixation pathway used for autotrophic
Fig. 8.18 The regulation of PEP carboxylase in growth and differs in some key enzymological
E. coli. The carboxylase is positively regulated reactions from the pathways discussed here.
les: 1, pyruvate by acetyl-CoA and negatively regulated by The reductive carboxylic acid pathway is
aspartate. descri bed in Section 13.1. 9.
201
glucose-6-P
I
I
.
PhosPhoerIPyruvate
(;r
0
pyruvate
11
~
~acetyI-CoA Fig
oxaloacetate ~ . citrate
attl
NADH
-r
malate
1
2~
an
po~
[cis-aconitate] car
~
fumarate i
isocitrate
FADH2-~7 att;
4~-NADPH
succinate cen
oxalosuccinate
Co
~6
5~ ene
IsuccinyI-CoA ,
Ia-ketoglutarate I dis
Fig. 8.19 The reductive citric acid pathway in fermenting bacteria. There are two "arms." One am
route oxidizes citrate to a-ketoglutarate. A second route reduces oxaloacetate to succinyl-SCoA. nu(
The enzyme a-ketoglutarate dehydrogenase is missing. The enzyme fumarate reductase replaces oce
succinate dehydrogenase. Enzymes: 1, citrate synthase; 2, 3, aconitase; 4, 5, isocitrate dehydrogenase; but
6, succinate thiokinase; 7, fumarate reductase; 8, fumarase; 9, malate dehydrogenase; 10, PEP we]
carboxylase; 11, pyruvate carboxylase.
the
ane
8.11 Chemistry of Some of the Because the oxygen in the carbonyl group in 1
Reactions in the Citric acid Cycle is electron attracting, there is a tendency to
pull electrons away from the C-H bond in the
This section presents a rational basis for 8.1
carbon adjacent to the carbonyl. This results
understanding the chemistry of some of the in the formation of an enol ate ion, which
key reactions in the citric acid cycle. Similar Ox
acts as a nucleophile (Fig. 8.21). The enolate (i.e,
reactions are seen in other pathways.
anion seeks electrophilic centers, (e.g., the Tht
carbon atoms in carbonyl groups). So acetyl- aCI<
8.11.1 Acetyl-CoA condensation CoA can be a nucleophile at its methyl end oth
reactions and attack other carbonyl groups, even other
acetyl-CoA molecules. In the formation of
Acetyl-CoA is a precursor for the biosynthesis citric acid, the methyl group of acetyl-CoA
of many different molecules besides citrate. attacks a carbonyl group in oxaloacetate to
These include lipids (Chapter 9) and various form citric acid. At the same time, the thioester
Fig.
fermentation end products (Chapter 14). The linkage to coenzyme A is hydrolyzed, driving
nuc,
reason why acetyl-CoA is so versatile is that the reaction to completion. Later, we will
disJ:
it undergoes condensations at both the methyl examine other pathways in which the methyl
and carboxyl end of the molecule. Condensa- carbon of acetyl-CoA attacks a carbonyl.
tions at both ends of acetyl-CoA can be under- Another result of the polarization of the
stood in terms of the chemistry of the polarized carbonyl group is that the carbon in the
carbonyl group. The electrons in the carbonyl carbonyl is electron deficient and subject to E
c
group are not shared equally by the carbon
and oxygen; rather, the oxygen is much more
"- c+=o-
electronegative than the carbon and pulls the /
electrons in the double bond closer to itself. Fig.
Fig. 8.20 The carbonyl group is polarized.
That is to say, the C=O group is polarized, an e
Electrons are attracted by the oxygen, leaving a deca
making the carbon slightly positive (Fig. 8.20).
partial positive charge on the carbon. clea.
202
H 1""0
I
\..)1
H-C-C-SCoA
IJ
H,
,
. [HJ.rt:SCoA - H-~J-S"'A]
enolate anion
'"
Fig. 8.21 The methylene carbon of acetyl-CoA can act as a nucleophile. Because of the electron-
attracting ability of the carbonyl group, a hydrogen dissociates from the methylene carbon, forming
an enolate anion, which resonates. Electrons can shift to the methylene carbon, which then seeks a
positive center, e.g., a carbonyl group. Because of this, acetyl-CoA will form covalent bonds to the
carbon of carbonyl groups in condensation reactions.
attack by nucleophiles that seek a positive phosphate pathway. When 6-phospho-

center (i.e., it is electrophilic). Thus acetyl- gluconate is oxidized by NADP+, a 3-keto
CoA undergoes condensations at the carboxyl intermediate is formed (Fig. 8.8). The de-
end in reactions in which the CoASH is carboxylation of ,B-ketocarboxylic acids is
displaced during a nucleophilic displacement relatively straightforward. A single enzyme
and the acetyl portion is transferred to the is required, and the cofactor requirements are
nucleophile (Fig. 8.22). For example, this met by Mn2+ or Mg2+. A ,B-decarboxylation is
occurs during fatty acid synthesis and during shown in Fig. 8.23. The ,B-keto group attracts
butanol fermentations (Chapters 9 and 14), as electrons, facilitating the breakage of the bond
well as in several other pathways. Because of holding the carboxyl group to the molecule.
the reactivity of acetyl-CoA at both the methyl Note the similarity to the aldolase reaction,
and carboxyl end, the molecule is widely used where the keto group facilitates the breakage
in building larger molecules. of a C-C bond that is beta to the keto group
(Fig. 8.5).
8.11.2 Decarboxylation reactions

Decarboxylation of a-ketoglutarate
Oxalosuccinate is a ,B-ketocarboxylic acid a-Ketoglutarate is an a-keto carboxylic acid,
(i.e., the carboxyl group is ,B to a keto group). and its decarboxylation is more complex
The decarboxylation of ,B-ketocarboxylic than that of a ,B-keto carboxylic acid.
acids occurs throughout metabolism. An- The mechanism is the same as for the
other ,B-decarboxylation occurs in the pentose decarboxylation of pyruvate, another a-keto
o
O'
II
CH3-C+-SCoA
n + :R-H .
II
CH3-C-R + HSCoA
~ "
Fig. 8.22 Acetyl group transfer. Because the carbonyl group in acetyl-CoA is polarized, it is subject to
nucleophilic attack. The result is that the acetyl group is transferred to the nucleophile and CoASH is
displaced.
H
I
-c-
I
c=o
I
Fig. 8.23 The decarboxylation of a ,B-keto carboxylic acid. The keto group attracts electrons causing
an electron shift and the breakage of the C-C bond holding the carboxyl group to the molecule. The
decarboxylation of oxalosuccinate is physiologically reversible. Notice the resemblance to the aldol
cleavage shown in Fig. 8.5.
203
carboxylic acid, and is described in more Carbon balance for the

detail in Section 14.10. Pyruvate and a- glyoxylate cycle
ketoglutarate dehydrogenases are similar in
The carbon balance is written below. The
that they can be separated into three compo-
net result of the glyoxylate cycle is the
nents (i.e., the TPP-containing decarboxylase,
condensation of two molecules of acetyl-CoA
the FAD-containing dihydrolipoyl dehydro-
to form succinate. Carbon balance for the
genase, and the diydrolipoyl transacety- glyoxylate pathway:
lase). Other a-ketocarboxylic acid dehydro-
genases exist (e.g., for the catabolism of C2 + C4 + C6
a-ketoacids derived from the degradation C6 + C4 + C2
of the branched-chain amino acids, leucine,
C2 + C2 + C4
isoleucine, and valine).
C2 + C2 + C4
8.12.1 Regulation of the

8.12 The Glyoxylate Cycle glyoxylate cycle
We now come to a second pathway central As Fig. 8.24 illustrates, isocitrate is at a

to the metabolism of acetyl-CoA: the glyoxy- branch point for both the citric acid cycle
late cycle, also called the glyoxylate bypass and the glyoxylate cycle. That is to say, it is
(Fig. 8.24). The glyoxylate cycle is required a substrate for both the isocitrate lyase and
by aerobic bacteria to grow on fatty acids the isocitrate dehydrogenase. What regulates
the fate of the isocitrate? In E. coli the
and acetate. (Plants and protozoa also have Fig. 8.24
isocitrate dehydrogenase activity is partially represent
the glyoxylate cycle. However, it is absent in
inactivated by phosphorylation when cells
animals.) The glyoxylate cycle resembles the
are grown on acetate.13 (The regulation of intermec
citric acid cycle except that it bypasses the two
enzyme activity by covalent modification is
decarboxylations in the citric acid cycle. For generallJ
discussed in Chapter 6, and other examples are
this reason the acetyl-CoA is not oxidized to via the d
listed in Table 6.1.) Acetate also induces the
C02. Examine the summary of the glyoxylate the phm
enzymes of the glyoxylate cycle. Therefore, in
cycle shown in Fig. 8.24. The glyoxylate cycle reactioTIi
the presence of acetate, isocitrate lyase activity
shares with the citric acid cycle the reactions increases while the isocitrate dehydrogenase
that synthesize isocitrate from acetyl-CoA. is partially inactivated. However, the Km for
The two pathways diverge at isocitrate. In the
8.13.1,
the isocitrate lyase is high relative to the phosph
glyoxylate cycle the isocitrate is cleaved to suc- concentrations of isocitrate. This means that
cinate and glyoxylate by the enzyme isocitrate
oxaloal
the isocitrate lyase requires a high intracellular
lyase (reaction 1). The glyoxylate condenses concentration of isocitrate. It is presumed
This reac
with acetyl-CoA to form malate, in a reaction that the partial inactivation of isocitrate
lation c:
catalyzed by malate synthase (reaction 2). The dehydrogenase increases the concentration of
shown ir
malate synthase reaction is of the same type as isocitrate, resulting in an increase in flux
and acc(
the citrate synthase (i.e., the methylene carbon through the glyoxylate cycle.
thesis in
of acetyl-CoA attacks the carbonyl group in
This enz:
glyoxylate). The reaction is driven to com-
portant I
pletion by the hydrolysis of the coenzyme A 8.13 Formation of (Section
thioester bond just as during citrate synthesis. Phosphoenolpyruvate IS an 1m
The malate replenishes the oxaloacetate, leav- phospho,
ing one succinate and NADH as the products. Organic acids such as lactate, pyruvate, ac- catalyzes
The cells incorporate the succinate into cell etate, succinate, malate, amino acids, and so phosphol
material by first oxidizing it to oxaloacetate on can be used for growth because pathways teria. Fo
from which phosphoenolpyruvate is synthe- exist to convert them to phosphoenolpyru- ferment !
sized (Section 8.12). vate, which is a precursor to the glycolytic zyme to I
204
acetyl-coA
~
CoASH
oxaloaeetate H20
~ ~
1
NADH H+ citrate
NAD+
yOOH
II
0
C,H)-C-SCoA
\ H20
[cis-aeonitate]
CH2
I
CHOH
I
COOH
...
malate
CoASH HP
\ /
2
\.
H-C='D'
I
COOH
gIyoxylate
\
COOH
I
I
HP
CH2
I
H-C-COOH
H~y-H
COOH ~OOH isocitrate
I
yH2 NADP+
"
yH2 :(NADPH +W
COOH Y
oxalosuccinate
~ succinate
ATP~"
~
/\ "CO
ADP + P; " 2
~, " a-ketoglutarate
.
CoASH : ..'
SUCClDyJ-C~_ __ _____ __ .-rNAD+
--
I
C02 C~SH NADH + W
Fig. 8.24 The glyoxylate cycle. Enzymes: 1, isocitrate lyase; 2, malate synthase. The dotted arrows
represent the reactions of the citric acid cycle that are bypassed.
intermediates. The phosphoenolpyruvate is oxaloacetate and then in other reactions re-

generally made in two ways. It can be made duce the oxaloacetate to succinate.14)
via the decarboxylation of oxaloacetate or via
the phosphorylation of pyruvate. These two
reactions are described next. 8.13.2 Formation of
phosphoenolpyruvate from pyruvate
8.13.1 Formation of Prokaryotes are able to synthesize phospho-
phosphoenopyruvate from enolpyruvate by phosphorylating pyruvate,
oxaloacetate instead of converting the pyruvate to oxaloac-
etate and then decarboxylating the oxaloac-
This reaction is an ATP-dependent decarboxy- etate. This is necessary for growth on pyru-
lation catalyzed by PEP carboxy kinase, as vate for those bacteria that cannot synthesize
shown in Fig. 8.25. The enzyme is widespread oxaloacetate from pyruvate. Prokaryotes that
and accounts for phosphoenolpyruvate syn-
thesis in both eukaryotes and prokaryotes. COOH
This enzyme, along with malic enzyme, is im- I COOH
c=o I
portant for growth on succinate and malate I
yH2
+ ATP
- c-o-@
II + ADP + C02
(Section 8.14). (Although PEP carboxykinase CH2
COOH
is an important enzyme for the synthesis of
Fig. 8.25 The PEP carboxykinase reaction. The
phosphoenolpyruvate from oxaloacetate, it
PEP carboxykinase generally operates in the
catalyzes the synthesis of oxaloacetate from
direction of PEP synthesis, although during
phosphoenolpyruvate in some anaerobic bac-
fermentation in anaerobes it can work in the
teria. For example, anaerobic bacteria that direction of oxaloacetate, which is subsequently
ferment glucose to succinate may use this en- reduced to the fermentation end product,
zyme to carboxylate phosphoenolpyruvate to succinate.
205
fall into the latter category do not have a gly- pyrophosphoryl group transfer reactions.)
oxylate pathway and also lack pyruvate car- One phosphate is removed by hydrolysis, leav-
boxylase. For example, E. coli does not have ing a phosphorylated enzyme. The phospho-
a glyoxylate cycle unless growing on acetate, rylated enzyme then donates the phosphoryl
and it has PEP carboxylase instead of pyru- group to pyruvate to form PEP. The reaction
vate carboxylase. Furthermore, there are strict catalyzed by the pyruvate-phosphate dikinase
anaerobes (e.g., methanogens and green sulfur is similar except that, instead of the phosphate
photosynthetic bacteria) that grow autotroph- being hydrolytically removed from the py-
ically converting C02 to acetyl-CoA, which rophosphorylated enzyme, it is transferred to
is carboxylated to pyruvate (Sections 13.1.2 inorganic phosphate to form pyrophosphate.
and 13.1.3). They do not have a glyoxylate The pyrophosphate is hydrolyzed by a py-
cycle and must phosphorylate the pyruvate to rophosphatase, pulling the reaction to comple-
form phosphoenolpyruvate. The phosphory- tion. The net result from both reactions is the
lation of pyruvate to phosphoenolpyruvate is same (i.e., the sum of the pyruvate-phosphate
described next. dikinase and pyrophosphatase reactions is the
same as the PEP synthetase reaction). In either
case, the synthesis of phosphoenolpyruvate
PEP synthetase and
from pyruvate requires the hydrolysis of two
pyruvate-phosphate dikinase reactions 01
phosphodiester bonds with a high free energy ac
Prokaryotes that convert pyruvate directly to of hydrolysis.
phosphoenolpyruvate use one of two enzymes.
They are PEP synthetase and pyruvate-
phosphate dikinase. These enzymes are found 8.14 Formation of Pyruvate
in prokaryotes and plants, but not in animals. from Malate
The reactions are illustrated in Fig. 8.26.
Bacteria and mitochondria can convert malate
PEP synthetase
pyruvate + H20 + ATP ) directly to pyruvate using the malic enzyme:
Fig. 8.27
Malic enzyme
PEP +AMP+ Pi carbohydr
L-malate + NAD+ ,
pyruvate-phosphate dikinase
'
4, PEP ca
pyruvate + ATP + Pi pyruvate + NADH + C02 phosphofr
PEP + AMP + PPi Bacteria and mitochondria actually possess

the glyox
pyrophosphatase reaction two malic enzymes, one specific for NAD+
) lyase. Re
PPi + H20 2Pi and the other for NADP+. The latter provides
acids and
NADPH for reductions that occur during
In the PEP synthetase reaction, a pyrophos- that enter
biosynthesis, such as the biosynthesis of
phoryl group is transferred from ATP to the late cycle.
fatty acids (Chapter 9). Malic enzyme, along
enzyme. (See Fig. 7.3 for a description of are event
with PEP carboxykinase (Section 8.13.1), is
intermedi
an important enzyme for growth on citric
the Emb(
acid cycle intermediates such as succinate or
E+ATPDJ.w . E-PP+AMP malate. E. coli mutants that lack both PEP
pentose I
Doudoro
H20 Pi carboxykinase and malic enzyme will not
intersect
Pi PPi grow on succinate or malate, although strains
lacking only one of these enzymes will grow.
E-P
E4yruvale
8 .16 S~
8.15 Summary of the Relationships
PEP
between the Pathways The neal
Fig. 8.26 The PEP synthetase and pyruvate- degradat
phosphate dikinase reactions. Enzymes: 1, PEP Figure 8.27 illustrates the relationship be- pathway
synthetase; 2, pyruvate-phosphate dikinase; 3, tween the different pathways discussed in this way. Tn
pyrophosphatase. chapter. Reactions 1 and 2 are key enzymes in glucose 1
206
IS.)
av-
ho-
r::;::::::::::l
glucose - - - - - ~ G6P :>6-P-gluconate ~,'
)ryl ~ ~"_ £Y \7 II
;£:
IOn F6P "'"RuMP
ase
H20
Pi
8
~
7 --'-" --,,-',' , ,'~ KDPG
I
late F BP ,,'PPP /,'

py- / / ED //
~ -~-
I to PGALD ~ ,,'
~?--
ate. ~
py- ptp ,,/
I
ple-
~ ~
the ~/"
5 4
pyruvate
late
the
:her ~tyl-COA - - -I fattyacidsI
{ate
asp~te.-
,
- - - - - - oxalo~tate-
/
-/~ /~ citrate
0( 2
two Y malate glyoxylate
'\
l [cis-aconitate ]
~rgy other amino
acids ( \
fumM~ l' isocitrate
\ . I
succm", oxalosuccinate
succinyl-CoA
A
, a: keogu
t I tara:-
t " co
,
. ~
,, ~ C~
'2,,
\~
llate ammo act~ S,
glutamate
_____ .. otheraminoacids
tetrapyrro les
ne:
Fig. 8.27 Relationship between the glyoxylate cycle, the citric acid cycle, and the major
carbohydrate pathways. Enzymes: 1, isocitrate lyase; 2, malate synthase; 3, pyruvate carboxylase;
4, PEP carboxylase; 5, PEP carboxykinase; 6, PEP synthetase or pyruvate phosphodikinase; 7,
phosphofructokinase; 8, fructose-1,6-bisphosphatase. Not shown is malic enzyme (Section 8.14).
;sess
the glyoxylate cycle. Reaction 1 is isocitrate duces two moles of NADH and two moles
AD+
lyase. Reaction 2 is malate synthase. Fatty of ATP. There is only one oxidation, and
rides
acids and acetate are converted to acetyl-CoA that is the oxidation of phosphoglyceralde-
lflng
; of that enters the citric acid cycle and the glyoxy- hyde to phosphoglycerate. An intermediate,
late cycle. Dicarboxylic acids and amino acids 1,3-bisphosphoglycerate, is formed, which
long
are eventually degraded to citric acid cycle donates a phosphoryl group to ADP in a
l), is
intermediates. Sugars can be catabolized via substrate-level phosphorylation. Since two
:itric
the Embden-Meyerhof-Parnas pathway, the phosphoglyceraldehydes are formed from one
te or
pentose phosphate pathway, or the Entner- glucose, two ATPs are made. The pathway
PEP
Doudoroff pathway. All the sugar pathways can be considered as two stages. Stage 1 gen-
not
intersect at phosphoglyceraldehyde. erates two phosphog]yceraldehydes. Stage 2
rams
'ow. is the oxidation of phosphoglyceraldehyde to
pyruvate. Glycolysis not only provides ATP,
8.16 Summary NADH, and pyruvate, but its intermediates
are used for biosynthesis in other pathways.
The nearly ubiquitous pathway for glucose This will become more evident in subsequent
degradation is the Embden-Meyerhof-Parnas chapters when we examine other metabolic
) pathway, also called the glycolytic path- pathways. The pathway cannot be re-
be-
l this way. The pathway oxidizes one mole of versed from pyruvate to glucose-6-phosphate
les m glucose to two moles of pyruvate, and pro- because of two irreversible reactions,
207
10. Selig, M., and P. Schonhet. 1994. Oxidation Pseudomonas stutzeri. Aerobic and nitrate res-
of organic compounds to C02 with sulfur or piration routes of carbohydrate catabolism.
thiosulfate as electron acceptor in the anaero- ]. Bacterial. 91:245-250.
bic hyperthermophilic archaea Thermoproteus 13 . L a P one, D ..C 1993 . Th e IsoCitrate de hy d ro-
tenax an d Pyro bacu Ium /s Ian d /cum procee d s via
" "
"
" "
genase phosphorylation cycle: Regulation and
tecltnCaCi
h "" " - I A rc.h M/cr%.b I 162..286-
d cyce. " "
enzymoIogy. fCIIB
"
. e. /oc" h em. 51 .. 1418

- .
"
294.
14. Podkovyrov, S. M., and.. j. G. Zeikus. 1993.
11
.
. Stewart,. V 1988 . N Itrate
" respiratIOn
"." In re I.a- P un "fication an d c h aractenzatlon 0 f p h osp h 0-
metabohsm".
'
tlOn to facultative" In Enterobactena.
eno Ipyruvate car b oxy k"Inase, a cata b0 I"IC CO 2-
Microbial. Rev. 52: 190-232. fixing enzyme, from AnaeroblOspmllum suc-
12. Spangler, W. j., and C. M. Gilmour. ciniciproducens. ]. Gen. Microbial. 139:223-
1966. Biochemistry of nitrate respiration in 228.

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8

Central Metabolic Pathways

The central metabolic pathways are those is incorporated into an acyl-phosphate

t~-- ATP, NADH

tory phosphorylation as explained in Chapter Sum: Glucose + 2ADP + 2Pj + 2NAD+

kinase reaction is 3-phosphoglycerate (3- glycolysis is coupled to the oxidation of phos-

polysaccharides, pentose phosphates,

.. serine, glycine, cysteine

lipids, amino acids, .. PEP aromatic amino acids,

Fig. 8.4 The fructose-l,6-bisphosphatase reaction

8.2 The Fate of NADH is written NADH, not NADH2. To under-

glucose and fructose do not support growth, glucose

C02 by pyruvate-ferredoxin oxidoreductase. ADP

ribulose-S-P (RuMP) CH200

Fig. 8.8 The oxidation-decarboxylation reactions. Enzymes: (1), glucose-6-phosphate dehydrogenase;

(Fig. 8.9). of C02 and one mole of phosphoglycer-

yHzOH yHzOH H-C=O

C=O - -- - -- 'r:. H-C=O ;H20H

Oxidative decarboxylation 3 glucose-6-P + 3 ribulose-S-P + 3C02

Isomerizations 3 ribulose-S-P + 2 xylulose-S-P + ribose-S-P

Transketolase xylulose-S-P + ribose-S-P + sedoheptulose-7-P + phosphoglyceraldehyde

Transaldolase sedoheptulose-7-P + phosphoglyceraldehyde + fructose-6-P + erythrose-4-P

Transketolase xylulose-S-P + erythrose-4-P + fructose-6-P + phosphoglyceraldehyde

Sum: glucose-6-P + phosphoglyceraldehyde + 3C02

8.6 The Entner-Doudoroff

The pathway diverges from the pentose phos-

COO COO. COO.

pathway is so common in the bacteria. gluconate may confer a competitive ad-

'f decarboxylation of pyruvate to acetyl-CoA.

attack by nucleophiles that seek a positive phosphate pathway. When 6-phospho-

8.11.2 Decarboxylation reactions

carboxylic acid, and is described in more Carbon balance for the

8.12.1 Regulation of the

We now come to a second pathway central As Fig. 8.24 illustrates, isocitrate is at a

intermediates. The phosphoenolpyruvate is oxaloacetate and then in other reactions re-

PEP + AMP + PPi Bacteria and mitochondria actually possess

late F BP ,,'PPP /,'

. e. /oc" h em. 51 .. 1418

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