362 SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP

Initially, the acetyl group is incorporated into citrate, an intermediate of the TCA
Synthases, such as citrate synthase,
catalyze condensation of two
Resumo do Capítulo 20 do Marks - O Cíclo do Ácido Tricarboxílico, ou
cycle (Fig. 20.3). As citrate progresses through the cycle to oxaloacetate, it is oxi-
organic molecules to form a car-
dized by four dehydrogenases (isocitrate dehydrogenase, "-ketoglutarate dehydro-
Cíclo de Krebbs, ou Cíclo do Ácido Cítrico
bon–carbon bond. Dehydrogenases, such as
genase, succinate dehydrogenase, and malate dehydrogenase), which transfer elec-
isocitrate dehydrogenase, are enzymes that
trons to NAD! or FAD. The isomerase aconitase rearranges electrons in citrate,
remove electron-containing hydrogen or
thereby forming isocitrate, to facilitate an electron transfer to NAD!.
hydride atoms from a substrate and transfer
Although no O2 is introduced into the TCA cycle, the two molecules of CO2 pro-
I. Introdução:
them to electron-accepting coenzymes, such
duced have more oxygen than the acetyl group. These oxygen atoms are ultimately
as NAD! or FAD. Aconitase is an isomerase,
A. O CAT é responsável por 2/3
an enzyme that catalyzes an internal
da produção
derived de energia
from the carbonyl da oxidação
group of acetyl de combustíveis
CoA, two molecules of water added by
B. Substrato: acetil CoA 2-
fumarase and citrate synthase, and the PO4 added to GDP.
rearrangement of atoms or electrons. In
C. Ocorre na mitocôndria The overall yield of energy-containing compounds from the TCA cycle is 3
aconitase, a hydroxyl group is being trans-
NADH, 1 FAD(2H), and 1 GTP. The high-energy phosphate bond of GTP is gener-
ferred from one carbon to another. An iron
D. Controlado pela taxa de produção
ated from da
cofactor in the enzyme facilitates this transfer. cadeia
substrate levelde transportecatalyzed
phosphorylation de elétrons e dathiokinase (suc-
by succinate
fosforilação oxidativa pelocinyl
mecanismo de feed-back
CoA synthetase). As the NADH and FAD(2H) are reoxidized in the electron
E. Função do cíclo: conser var atransport
energia chain,gerada na oxidação
approximately 2.5 ATP arede acetilforCoA
generated each NADH, and 1.5 ATP
II. Reações do CAT:
O
CH3C SCoA
Acetyl CoA
–
COO
CoASH
C O COO–
citrate synthase
CH2 CH2
H2O
malate –
COO HO C COO–
dehydrogenase
Oxaloacetate CH2
– aconitase
COO– COO
NADH Citrate COO–
HO CH + + H+
NAD
CH2
CH2
H C COO–
COO–
Malate HO C H
–
electron COO
H2O ATP transport Isocitrate
fumarase chain
–
NAD+
COO Oxidative
CO2
phosphorylation NADH + H+
HC H2O O2
isocitrate
CH COO– dehydrogenase
COO– CH2
Fumarate
FADH(2H) NADH CH2
+ H+ C O
FAD
COO– NAD+
COO–
succinate CH2 COO– α –Ketoglutarate
dehydrogenase
CoASH CH2
CH2 CO2
–
COO CH2 CoASH α –ketoglutarate
Succinate GDP C O dehydrogenase
+ Pi
˜

succinate SCoA
thiokinase
GTP
Succinyl CoA

A. Formação e oxidação de isocitrato:

Fig. 20.3. Reactions of the TCA cycle. The oxidation-reduction enzymes and coenzymes are shown in blue. Entry of the two carbons of acetyl
CoA into the TCA cycle are indicated with blue dashed boxes. The carbons released as CO2 are shown with black dashed boxes.
1. Oxalacetato e o grupo acetil do acetil CoA se condensam para formar
citrato
2. Como o oxalacetato é regenerado ao final do cíclo, ele não é considerado
substrato e nem fonte de elétrons e de carbonos
3. O grupo hidroxila do citrato é movido para o carbono 2 para ser oxidado para
cetona pela enzima aconitase
 4. O isocitrato é oxidado pela ação da enzima isocitrato desidrogenase a α-
cetoglutarato e um CO2 é liberado
5. NAD+ --> NADH + H+
B. α- cetoglutarato a succinil CoA
1. Reação catalisada pela enzima α- cetoglutarato desidrogenase
2. Há liberação de um grupo carboxila
CHAPTER 20 / TRICARBOXYLIC ACID CYCLE 365
na forma de CO2 e a cetona é oxidada a
ácido carboxílico, que reage com CoASH para formar succinil CoA
3. NAD+ --> NADH + H+
The claim that succinate oxidation
COO –
4. O succinil CoA couldtem uma
produce ligação
energy withouttioester,
oxy- ligação muito energética
CH2 C. Geração de GTP (Guanosina Trifosfato):
α-Ketoglutarate
gen is wrong. It was probably
based on the fact that succinate is oxidized
CH2 1. Energia da ligação tioester é usada para gerar GTP a partir de GDP
to fumarate by the donation of electrons to
C O 2. Reação: FAD.fosforilação
However, ATP can a only
nívelbedo substrato, ou seja, não usa oxigênio molecular
generated

H O
COO
–
para fosforilar, ou seja, NÃO é fosforilação
from this process when these electrons are oxidativa
donated to oxygen in the electron transport
C NH2 3. A quantidade de energia
chain. The energy generatedarmazenada
by the electron no GTP é equivalente à do ATP
+ H+ D. Oxidaçãotransport
do succinato a oxaloacetato:
chain is used for ATP synthesis in
1. Há 2 pares de elétrons sobrando para reagir no succinato
the process of oxidative phosphorylation.
After the covalently bound FAD(2H) is oxi-
H
2. Succinato é oxidado
dized back to FAD by athefumarato e FAD --> FAD(2H)
electron transport
3. A dupla ligação
chain, succinate dodehydrogenase
fumaratocan é quebrada
oxidize por um grupo OH- e um próton da
he alcohol group (C—OH) is oxi- another succinate molecule.
água, convertendo o fumarato a malato
D! as the hydride ion. Subsequent
4. O grupo OH- do malato é oxidado a cetona pela doação de elétrons a NAD+ e
arge. The H of the OH group dis-
cceptor, is reduced. oxalaxetato é regenerado
III. Coenzimas do CAT:
A. FAD e NAD+
pts a pair of electrons as the
1. São coenzimas aceptoras de elétrons
opposite the positively-charged
2. FAD,
in the oxidation of alcohols to a certinha:
ydrogenase. The nicotinamide a) Aceita elétrons de substratos ligados à mesma enzima
nd the alcoholic hydrogen b) is Aceita 1 elétron de cada vez e forma um intermediario “metade-
oton, H!.
e very reactive, and FADH can
reduzido”, o FADH, que é muito reativo. Portanto, para não perder o elétron,
tiation of chain reactions. As a a molécula de FADH deve manter-se fortemente ligada à sua enzima -->
etimes covalently, attached to não muda de enzima quando recebe elétrons --> certinha
o another group bound on the
ny functional groups on amino Succinate Fumarate
-bound FAD varies greatly and
ontrast, NAD! and NADH are
His – FAD

rgy metabolism that FAD(2H)

nzyme. Free NAD! binds to a Fe – S
then released into the medium
enase. Consequently, oxidative Inner CoQ
, and do not generate NADH mitochondrial ETC acceptor
membrane
ort chain. The regulation of the CoQH2
the NADH/NAD! ratio is part
oxidation to the rate of ATP Succinate
dehydrogenase

Fig. 20.6.em
c) É usado Succinate
reaçõesdehydrogenase
de formaçãocontainsde duplas ligações (succinato a fumarato)
covalently bound FAD. As a consequence, suc-
d) Não tem
cinate ação regulatória
dehydrogenase and similar em função da ligação com a enzima
flavopro-
ctions through the formation of teins reside in the inner mitochondrial mem-
and an acyl group (e.g., acetyl brane where they can directly transfer
electrons into the electron transport chain. The
electrons are transferred from the covalently
me, and NAD! is its promiscuous bound FAD to an Fe-S complex on the
rom a substrate that is bound to enzyme, and then to coenzyme Q in the elec-
onates these without leaving that tron transport chain (see Chapter 21). Thus,
enzyme. NAD!, conversely, may FAD does not have to dissociate from the
and leaves the enzyme immedi- enzyme to transfer its electrons. All the other
d to a different dehydrogenase, enzymes of the TCA cycle are found in the
 physiological roles in the cell. FAD is able to accept single electrons (H•), and
forms a half-reduced single electron intermediate (Fig. 20.4). It thus participates in
reactions in which single electrons are transferred independently from two different
atoms, which occurs in double bond formation (e.g., succinate to fumarate) and
disulfide bond formation (e.g., lipoate to lipoate disulfide in the !-ketoglutarate

1 e–, H+
H H
O O– O
CH3 N Single CH3 N+ Single CH3 N
NH electron • NH electron NH
– + – +
O 1e , H O 1e , H O
CH3 N N CH3 N N CH3 N N
CH2 – + R R H
1e , H
HCOH Riboflavin FADH • FADH2
FMN
HCOH (half reduced semiquinone) (fully reduced)

HCOH
CH2
FAD
O
–
O P O
O
–
NH2
O P O
N N
O
CH2 O N N

H H
H H
OH OH
Flavin adenine dinucleotide (FAD) and
flavin mononucleotide (FMN)

Fig. 20.4. One-electron steps in the reduction of FAD. When FAD and FMN accept single electrons, they are converted to the half-reduced semi-
3. NAD+,
quinone, a putinha:
a semistable free radical form. They can also accept two electrons to form the fully reduced form, FADH . However, in most dehydro- 2
genases, FADH is never formed. Instead, the first electron is shared with a group on the protein as the next electron is transferred. Therefore, in
a)overall
this text, Aceita
2
acceptance pares de elétrons
of two electrons by FAD has beendedenoted
substrato ligado
by the more general a qualquer
abbreviation, FAD(2H). enzima
b) Muda de enzima quanto recebe elétrons --> putinha CHAPTER 20 / TRICARBOXYLIC ACID

c) Usado na oxidação de álcool a cetona

– The claim that su
COO COO– could produce en
CH2 CH2 gen is wrong.
Isocitrate CO2 α-Ketoglutarate
H C COO
– based on the fact that suc
isocitrate CH2
to fumarate by the donatio
H O C H dehydrogenase
C O
••

FAD. However, ATP can o

– – from this process when th
COO
O H O COO
donated to oxygen in the e
C NH2 C NH2 chain. The energy generate
+ H+ transport chain is used for
+
N N the process of oxidative
R R After the covalently bound
NAD
+
NADH dized back to FAD by the e
chain, succinate dehydroge
d) Ação
Fig. regulatória:
20.5. Oxidation and decarboxylation of isocitrate. The alcohol group (C—OH) is oxi- another succinate molecule
dized to a ketone, with the C—H electrons donated to NAD! as the hydride ion. Subsequent
(1)electron
NAD+ liga-se a uma desidrogenase e é reduzido a NADH
shifts in the pyridine ring remove the positive charge. The H of the OH group dis-
(2)sociates
NADH intoéwater
liberado e pode
as a proton, ligar-se
H!. NAD ! a outra
, the electron desidrogenase
acceptor, is reduced. para inibí-la
(3) Portanto, a razão NAD+/NADH é responsável pela regulação de
enzimas oxidativas. NADH não é regenerado mais rapidamente do que
é consumido
dehydrogenase pela cadeia
reaction). de transporte
In contrast, NAD! accepts de elétrons
a pair of electrons as the
"
(4)hydride
É parteion (H
do ),mecanismo
which is attracted
quetoregula
the carbon opposite thedepositively-charged
a produção energia pela
pyridine ring (Fig. 20.5). This occurs, for example, in the oxidation of alcohols to
quantidade de ATP presente
ketones by malate dehydrogenase and isocitrate dehydrogenase. The nicotinamide
B. Os complexos α- cetoácido-desidrogenase:
ring accepts a hydride ion from the C-H bond, and the alcoholic hydrogen is
released into the medium as a positively charged proton, H!.
1. Tiaminapirofosfato:
The free radical, single-electron forms of FAD are very reactive, and FADH can
a) Quebra
lose its ligação C-C próximas
electron through exposure toàwater
cetonas α-reactions.
--> ageofnochain
or the initiation cetoglutarato
As a
consequence, FAD must remain very tightly, sometimes covalently, attached to
its enzyme while it accepts and transfers electrons to another group bound on the
enzyme (Fig 20.6). Because FAD interacts with many functional groups on amino Succinate F
acid side chains in the active site, the E0# for enzyme-bound FAD varies greatly and
can be greater or much less than that of NAD!. In contrast, NAD! and NADH are
His – FAD
more like substrate and product than coenzymes.
NADH plays a regulatory role in balancing energy metabolism that FAD(2H)
cannot because FAD(2H) remains attached to its enzyme. Free NAD! binds to a Fe – S
dehydrogenase and is reduced to NADH, which is then released into the medium
where it can bind and inhibit a different dehydrogenase. Consequently, oxidative Inner
enzymes are controlled by the NADH/NAD! ratio, and do not generate NADH mitochondrial
membrane
faster than it can be reoxidized in the electron transport chain. The regulation of the
TCA cycle and other pathways of fuel oxidation by the NADH/NAD! ratio is part
 CoA, succinyl CoA) (Fig. 20.7). The com
precursor, pantothenate, is shown in Fig
typical oxygen ester bond because S, unli
ticipate in resonance formations. One of
δ COO– chemistry is that the carbonyl carbon, th
SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP γ CH2 group in a CoA thioester can be activated
tions (e.g., in the citrate synthase reaction
β CH2
for condensation with oxaloacetate, see
The E0! values were calculated
α C in aOtest tube under standard conditions. When
quence is that the thioester bond is a hig
FAD is bound to an enzyme, as it is in the "-keto acid dehydrogenase com-
COO
–
0 #G0$ of hydrolysis (approximately–13 kc
plexes, amino acid side chains can alter its E ! value. Thus, the transfer of elec-
trons from the bound FAD(2H) toαNAD # The energy
in dihydrolipoyl dehydrogenase is actually
–Ketoglutarate ener- from cleavage of the high
getically favorable. and acetyl CoA is used in two different w
NAD+ Thiamine– P P CoA thioester bond is cleaved by succina
Lipoate for activating an enzyme-bound phosph
Arsenic poisoning is caused by the CoASH FAD
a vitamin precursor. Lipoate is attached αto–ketoglutarate
the transacylase enzyme 20.7B). In contrast,
through its when the thioester bo
presence of a large number of dif- synthase reaction, the energy is released,
ferent arsenious compounds that carboxyl group, which is covalently CO2 bound to the terminal
dehydrogenase complex -NH of a lysine in the
2
of –7.7
protein (Fig. 20.10). At its functional end, lipoate contains a disulfide group that kcal/mole. The large negative #G
ctive metabolic inhibitors. Acute
accepts electrons when it binds NADH TCA cycle
the acyl fragment of "-ketoglutarate. It can thus act going in the forward direction
al or intentional arsenic poisoning
+ H+
high doses and involves arsenate like a long flexible -CH2- arm of the enzyme that reaches over to the decarboxylase
δ COO– C.siteThe !-Ketoacid Dehydrogen
and arsenite (AsO2&). Arsenite, to pick up the acyl fragment from thiamine and transfer it to the active contain-
10 times more toxic than arsenate, ing bound CoASH. It then swings over γ CHto2 dihydrolipoyl dehydrogenase to transfer
The !-ketoglutarate dehydrogenase com
o neighboring sulfhydryl groups, electrons from the lipoyl sulfhydrylβgroups CH2 to FAD. similar !-keto acid dehydrogenase comple
those in dihydrolipoate and in
cysteine pairs (vicinal) found in "-
O the pyruvate dehydrogenase complex, an
3. FAD AND DIHYDROLIPOYL DEHYDROGENASE α C SCoA acid dehydrogenase complex. Each of the
d dehydrogenase complexes and in
dehydrogenase. Arsenate weakly Succinyl CoA
FAD on dihydrolipoyl dehydrogenase accepts electrons from the lipoyl sulfhydryl keto acid structure. In the sequence of rea
enzymatic reactions involving phos- groups= and transfers them to bound NAD #
. FAD thus accepts ketoacid
transfers iselec-
and cetoglutarato,decarboxylated (i.e., releases t
b) Tiamina
ncluding the enzyme glyceraldehyde
vitamina B1
Fig.-->
20.8.falta causa
Oxidative acúmulo
decarboxylation de !-α-
of keto group o to the level of a
is oxidized
trons without leaving its binding site
ketoglutarate. Theon!-ketoglutarate
the enzyme. dehydroge-
The direction of the reaction
ydrogenase in glycolysis (see Chap- CoASH to form
its an acyl CoA thioester (e
que barra o CAT
is favored e, portanto,
by interactions
nase of FAD
complex a oxidizes
quebra
with groups aeróbica dasuc-
on the enzyme,
!-ketoglutarate to glicose
which change
cleavage and oxida-!-ketoacid dehydrogenase
All of the
Thus both aerobic and anaerobic ATP
2. Lipoato:reduction potential andcinyl
on can be inhibited. The low doses
by the
CoA.overall release group
The carboxyl of energy from as
is released
composed of multiple subunits of thr
tion of "-ketoglutarate.CO2. The keto group on the !-carbon is oxi-
a) Coenzima encontrada
ic compounds found in water sup- somente nos complexos
dized, and then forms the acyl CoA thioester,α- cetoácido-
(Fig. 20.9). E1 is an !-ketoacid de
e a major public health concern, but
succinyl CoA. The !, ", %, and & on succinyl pyrophosphate (TPP); it cleaves off the
desidrogenase
ciated with increased risk of cancer
III. ENERGETICS CoA OF THE
refer toTCA CYCLE
the sequence of atoms in !-ketog- a transacylase containing lipoate; it tran
han direct toxicity.
b) Não requer precursor
Like all metabolic vitamínico
lutarate.
pathways,
from thiamine to CoASH. E3 is dihydr
the TCA cycle operates with an overall net negative
O 3. FAD ligado
$G a0 diidrolipol
! (Fig desidrogenase:
20.11). The conversion of substrates to products is, therefore, energeti-
CH2 CH2 C a) FAD recebe elétrons de lipol
cally favorable. However, somesulfidril e os transfere
of the reactions, paradehydrogenase
such as the malate NAD+
reaction,
IV. Bioenergética do CAT: have a positive value.
CH CH2 CH2 N lysine–
H transacylase
S A. ∆Gº do CAT é negativo, portanto, o ciclo é espontâneo
enzyme

Lipoamide Acetyl CoA

(oxidized) CoA
Oxaloacetate
TPP–intermediate –7.7 kcal
NADH + H+ Citrate
NAD+
O +7.1 kcal +1.5 kcal
CH2 CH2 C Malate Isocitrate
CH CH2 CH2 N lysine–
H transacylase
S enzyme NAD+
O 0 kcal –5.3 kcal
C H2O NADH + H+

CH2 CO2

CH2 Fumarate α–Ketoglutarate

COO
–
0 kcal –8 kcal NAD+
FAD(2H) –0.7 kcal CoA NADH + H+
FAD Succinate CoA
10. Function of lipoate. Lipoate is CO2
Succinyl
to the %-amino group on the lysine
CoA
n of the tranacylase enzyme (E2). The GTP Pi GDP
lipoate disulfide formB.is reduced
Eficiência
as it do CAT:
the acyl group from1.thiamine É um processo
Fig. 20.11.extremamente
Approximate $G0! values eficiente. A combustão
for the reactions de 1given
in the TCA cycle, molfordetheacetil
for- CoA
sphate (TPP) attached to E1. The ward direction. The reactions with large negative $G0! values are shown in blue. The stan-
em um calorímetro
shown is for the "-ketoglutarate fornece
dard free energy ($G 0 228kcal
!) refers to e ochange
the free energy CAT for
fornece,
conversionem forma
of 1 mole de NAD,
of substrate
genase complex. FAD2H etoGTP,
1 mole207kcal (aprox.
of product under 90%
standard do total
conditions possível)
(see Chapter 19).
2. Reações reversíveis e irreversíveis termo ou cineticamente:
 a) Reações com ∆Gº bastante negativo e, portanto, extremamente
favoráveis. Essas reações são irreversíveis pois a quantidade de produtos
não é grande o suficiente para prevalecer sobre o ∆Gº e as enzimas
envolvidas catalisam a reação reversa muito lentamente. Essas reações
são responsáveis pelo ∆Gº< 0 do CAT e fazem com que o ciclo ande para
frente.
(1) Oxaloacetato/acetil CoA --> citrato
(2) Isocitrato --> α- cetoglutarato
(3) α- cetoglutarato --> succinil CoA
b) Reações com ∆Gº> 0, reversíveis termo e cineticamente
(1) Citrato <--> isocitrato: a enzima aconitase é rápida nos dois sentidos
da reação. Como a concentração de citrato é 20 vezes a concentração
de isocitrato, a reação ocorre mais no sentido do ciclo. O excesso de
citrato pode ser transportado para o citosol, onde dá origem a acetil
CoA
(2) Malato <--> oxaloacetato: há acumulo de malato, o que é influenciado
pela razão NADH/NAD+. Quando em jejum, o essa razão aumenta em
função da oxidação de ácidos graxos e, portanto, há acumulo maior de
malato, que é transportado para o citosol e sofre neoglicogênese
(fígado)
V. Regulação do CAT:
A. A taxa de CAT é regulada para manter a homeostase de ATP
B. CAT
370
é regulado principalmente em função da taxa da cadeia de transporte de
SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP
elétrons (regulada pela razão ATP/ADP)
Fuel oxidation

Acetyl CoA
CoA

Oxaloacetate – Citrate
Citrate NADH
H+ + NADH
citrate NAD+
– NADH synthase E
malate H+
NAD+ dehydrogenase T
H+
Malate Isocitrate O2 C
Isocitrate H2O
dehydrogenase
NAD+ ADP + Pi
+ ADP
– NADH
NADH + H+ ATP
H2O + Ca2+
CO2

Fumarate α-ketoglutarate
dehydrogenase α – Ketoglutarate
electron FAD(2H) CoA
– NADH
transport NAD+
FAD + Ca2+
chain Succinate NADH + H+
Succinyl CoA CO2
CoA
GTP Pi GDP

C. Na célula, a concentração de derivados de adenina (AMP, ADP, ATP) e de NAD

Fig. 20.12. Major regulatory interactions in the TCA cycle. The rate of ATP hydrolysis controls the rate of ATP synthesis, which controls the
rate of NADH oxidation in the electron transport chain (ETC). All NADH and FAD(2H) produced by the cycle donate electrons to this chain
(NAD+, NADH)
(shown on the sãooxidation
right). Thus, relativamente constantes.
of acetyl CoA in the Dessa
TCA cycle can go only as fastmaneira, uma
as electrons from NADHutlização
enter the electron transport
chain, which is controlled by the ATP and ADP content of the cells. The ADP and NADH concentrations feed information on the rate of oxida-
maior de ATP resulta em uma pequena redução na concentração de ATP e em um
tive phosphorylation back to the TCA cycle. Isocitrate dehydrogenase (DH), !-ketoglutarate dehydrogenase (DH), and malate dehydrogenase
pequeno aumento
(DH) are inhibited daNADH
by increased de ADP. "
concentration. The NADH/NAD ratio changes the concentration of oxaloacetate. Citrate is a product
2"
inhibitor of citrate synthase. ADP is an allosteric activator of isocitrate dehydrogenase. During muscular contraction, increased Ca concentra-
1.
tions“Mensageiros”
activate isocitrate DH and que acusam
!-ketoglutarate sobre taxa
dehydrogenase (as well de utilização
as pyruvate de ATP:
dehydrogenase).
a) O estado fosforilativo do ATP, refletido na razão ATP/ADP
oxidative pathways respond so rapidly to increased ATP demand that the ATP con-
centration does not significantly change.

A. Regulation of Citrate Synthase

 b) Estado reduzido do NAD+, refletido na razão NADH/NAD+
D. Regulação da citrato sintase:
1. Não tem reguladores alostéricos
2. Taxa é regulada principalmente pela concentração de oxaloacetato
(substrato) e de citrato (produto)
E. Regulação alostérica da isocitrato desidrogenase:
1. Regulação de vias metabólicas SEMPRE ocorre na enzima que catalisa a
reação mais lenta da via
2. Isocitrato desidrogenase é ativada alosetercamente por ADP e inibida por
NADH. É tambémCHAPTER
ativada por Ca2+
20 / TRICARBOXYLIC ACID CYCLE 371

pathways if product is not needed. Citrate synthase, A

e TCA cycle, is a simple enzyme that has no allosteric +ADP, K m
principally by the concentration of oxaloacetate, its sub- 0.1 mM
citrate, a product inhibitor, competitive with oxaloac-
alate-oxaloacetate equilibrium favors malate, so the v No ADP
very low inside the mitochondrion, and is below the K m 0.5 mM
A.4) of citrate synthase. When the NADH/NAD! ratio
etate to malate increases. When isocitrate dehydroge-
ation of citrate decreases, thus relieving the product
Thus, both increased oxaloacetate and decreased citrate
[Isocitrate]
citrate synthase to conditions established by the elec-
tive phosphorylation. In the liver, the NADH/NAD!
acetyl CoA enters the TCA cycle or goes into the alter- B
synthesis.

n of Isocitrate Dehydrogenase 6 fold

activation
n be made about regulation of metabolic pathways is v
that catalyzes the rate-limiting (slowest) step in a
citrate dehydrogenase is considered one of the rate-
le, and is allosterically activated by ADP and inhib- No ADP
n the absence of ADP, the enzyme exhibits positive
ds to one subunit, other subunits are converted to an [ADP]
pter 9, section III.A on allosteric enzymes). In the
bunits are in their active conformation, and isocitrate
ntly, the Km,app (the S0.5) shifts to a much lower value. C
isocitrate found in the mitochondrial matrix, a small
f ADP can produce a large change in the rate of the
ion. Small changes in the concentration of the prod-
trate, NAD!, also affect the rate of the enzyme more v
c enzyme.

oglutarate Dehydrogenase
enase complex, although not an allosteric enzyme, is
d succinyl CoA, and may also be inhibited by GTP (see [NADH]
glutarate dehydrogenase and isocitrate dehydrogenase Fig. 20.13. Allosteric regulation of isocitrate
the relative levels ofF.ADP
Regulação
and hence the α-
darate cetoglutarato
at which dehydrogenasedesidrogenase:
(ICDH). Isocitrate dehydroge-
transport. Both of these enzymes are also activated by nase has eight subunits, and two active sites.
scle, and possibly other 1. Inibida
muscle tissues,por NADH,ofsuccinil
the release CoA
Isocitrate, e !GTP
NAD , and NADH bind in the
2. Ativada
culum during muscle contraction por an
may provide Ca2+
addi- active site; ADP and Ca2! are activators and
mes when ATP isVI. being de acetil CoA: bind to separate allosteric sites. A. A graph of
rapidly hydrolyzed.
Precursores velocity versus isocitrate concentration shows
Cycle IntermediatesA. Fontes de acetil CoA: positive cooperativity (sigmoid curve) in the
serves two functions: it 1. β-that
ensures oxidação
NADH is de ácidosabsence
gener- graxosof ADP. The allosteric activator ADP
changes the curve into one closer to a rectan-
ATP homeostasis and it regulates the concentration of gularcetônicos
hyperbola, and
2. Degeneração dos corpos
example, in the liver, a decreased rate of isocitrate
β- decreases the Km (S0.5)
hidroxibutirato
for isocitrate. B. The allosteric activation by
e acetoacetato
e concentration, which stimulates citrate efflux to the ADP is not an all-or-nothing response. The
ry interactions occur in the TCA cycle, in addition to extent of activation by ADP depends on its
ontrol the levels of TCA intermediates and their flux concentration. C. Increases in the concentra-
TCA cycle. tion of product, NADH, decrease the velocity
of the enzyme through effects on the allosteric
activation.
 1. STRUCTURE OF PDC
PDC belongs to the "-ketoacid dehydrogenase complex family and, thus, shares
structural and catalytic features with the "-ketoglutarate dehydrogenase complex and
the branched chain "-ketoacid dehydrogenase complex (Fig. 20.15). It contains the
same three basic types of catalytic subunits: (1) pyruvate decarboxylase subunits that
3. Pode
bind ser formado a partir
thiamine-pyrophosphate de transacetylase
(E1); (2) acetato, que podethat
subunits virbind
da lipoate
dieta(Eou
2), da oxidação de
etanol
and (3) dihyrolipoyl dehydrogenase subunits that bind FAD (E3) (see Fig. 20.9).
Although the E1 and E2 enzymes in PDC are relatively specific for pyruvate, the same
4. Pode vir da glicose, alanina e serina, que dão piruvato, que é oxidado a acetil
dihydrolipoyl dehydrogenase participates in all of the "-ketoacid dehydrogenase
CoA pela complexo piruvato desidrogenase
5. Pode vir da leucina e da isoleucina, que também são oxidadas a acetil CoA
O COO–
+
CH3 H C H3N C H

CH2 O H C OH O CH3

CH2 C OH HO C H C OH The amino acid,

O alanine
C COO– CH2 6 CH2 H C OH C O

ruvate CH2 C O H C OH CH3 CH2OH

Thiamine – P P COOH CH3 CH2OH CH3

Pyruvate
Lipoate
FAD The fatty acid, The ketone body, The sugar, Ethanol
palmitate acetoacetate glucose
Pyruvate
dehydrogenase
complex
O
O
C ~ SCoA CH3 C SCoA

tyl CoA
Fig. 20.14. Origin of the acetyl group from various fuels. Acetyl CoA is derived from the
B. O complexo
ate dehydrogenase complex oxidation ofpiruvato desidrogenase
fuels. The portions (PDC):bodies, glucose, pyruvate, the amino
of fatty acids, ketone
1. Função:
oxidation of the "-ketoacid oxida
acid alanine, piruvato
and ethanol that areaconverted
acetiltoCoA, portanto,
the acetyl ligando
group of acetyl a shown
CoA are glicólose
in ao CAT
CoA. blue.
2. Estrutura:
a) Da família da α- cetoglutarato desidrogenase
b) Tem 3 sítios catalíticos:
(1) Piruvato descarboxilase que liga a tiamina-pirofosfato (E1) CHAPTER 20 / TRICARBOXYLIC ACID CYCLE 373

(2) Transacetilase queIn addition

complexes. ligatoatheselipoato (E2)the PDC complex contains one
three types of subunits,
additional catalytic subunit, protein X, which is a transacetylase. Each functional
Deficiencies of the pyruvate dehy-
drogenase complex (PDC) are

(3) Diidrolipoil desidrogenase

component of the PDC complexque liga
is present a FAD
in multiple (E3)
copies (e.g., bovine heart PDC
has 30 subunits of E , 60 subunits of E , and 6 subunits each of E and X). The E
among the most common inher-
ited diseases leading to lacticacidemia and,

c) Além desses sítios, existe umofsítio catalítico

types of subunits,!adicional, a proteína X, que é
1 2 3 1
like pyruvate carboxylase deficiency, are
enzyme is itself a tetramer two different and ".
grouped into the category of Leigh’s disease.

uma transacetilase 2. REGULATION OF PDC

In its severe form, PDC deficiency presents
with overwhelming lactic acidosis at birth,
with death in the neonatal period. In a sec-
3. Regulação do PDC: PDC activity is controlled principally through phosphorylation by pyruvate dehy-
drogenase kinase, which inhibits the enzyme, and dephosphorylation by pyruvate
ond form of presentation, the lactic acade-
mia is moderate, but there is profound psy-
a) Ocorre principalmente
nase kinase andpela fosforliação daregulatory
piruvato
dehydrogenase phosphatase, which activates it (Fig. 20.16). Pyruvate dehydroge-
pyruvate dehydrogenase phosphatase are desidrogenase
subunits within
chomotor retardation with increasing age. In
many cases, concomitant damage to the

quinase, que inibefromPDC, e desfosforilação pela piruvato

the PDC complex and act only on the complex. PDC kinase transfers a phosphate
ATP to specific serine hydroxyl (ser-OH) groups on pyruvate decarboxylase desidrogenase
brain stem and basal ganglia lead to death in
infancy. The neurological symptoms arise

fosfatase, que ativa PDC.

lation of just
1
Essas
one serine on the PDC enzimas são unidades
E ! subunit can decrease
1 its activity by over reguladoras de PDC
(E ). PDC phosphatase removes these phosphate groups by hydrolysis. Phosphory- because the brain has a very limited ability
to use fatty acids as a fuel, and is, therefore,

e agem somente nesse complexo. PDC quinase transfere fosfato de ATP

99%. PDC kinase is present in complexes as tissue-specific isozymes that vary in dependent on glucose metabolism for its
their regulatory properties. energy supply.
PDC kinase is, itself, inhibited by ADP and pyruvate. Thus, when rapid ATP uti- The most common PDC genetic defects
para E1 e PDC fosfatase
lization results in an increase of ADP, orfosfato
transfere when activation ofdeglycolysis
PDCincreases
por hidrólise. PDC are in the gene for the ! subunit of E1. The E1
!-gene is X-linked. Because of its impor-
pyruvate levels, PDC kinase is inhibited, and PDC remains in an active, nonphos-
quinase é inibida phorylated
por ADP form. PDC e piruvato.
phosphatase requires Portanto,
Ca for full activity.quando
2#
In the heart, há excesso de ADP
2# tance in central nervous system metabolism,
pyruvate dehydrogenase deficiency is a
increased intramitochondrial Ca during rapid contraction activates the phos-
e de piruvato, PDCphatase,
continua
thereby increasing ativo.
the amount of PDC fosforilase
active, nonphosphorylated PDC. requer Ca2+ para
problem in both males and females, even if
the female is a carrier. For this reason, it is
PDC is also regulated through inhibition by its products, acetyl CoA and NADH.
atividade máxima This inhibition is stronger than regular product inhibition because their binding to
classified as an X-linked dominant disorder.

Pi
PDC
inactive

ADP

ADP –
Pyruvate –
kinase phosphatase + Ca2+
Acetyl CoA +
NADH +

ATP Pi

PDC
active

+ –
Pyruvate Acetyl CoA

CoASH CO2

NAD+ NADH
+ –

Fig. 20.16. Regulation of pyruvate dehydrogenase complex (PDC). PDC kinase, a subunit of
the enzyme, phosphorylates PDC at a specific serine residue, thereby converting PDC to an
inactive form. The kinase is inhibited by ADP and pyruvate. PDC phosphatase, another sub-
unit of the enzyme, removes the phosphate, thereby activating PDC. The phosphatase is acti-
vated by Ca2#. When the substrates, pyruvate and CoASH, are bound to PDC, the kinase
activity is inhibited and PDC is active. When the products acetyl CoA and NADH bind to
PDC, the kinase activity is stimulated, and the enzyme is phosphorylated to the inactive form.
E1 and the kinase exist as tissue-specific isozymes with overlapping tissue specificity, and
somewhat different regulatory properties.
 VI. TCA CYCLE INTERMEDIATES AND ANAPLEROTIC
REACTIONS
A. TCA Cycle Intermediates are Precursors for
Biosynthetic Pathways
b) Ocorre
vate, citrate, !-ketoglutarate também
The intermediates por
of the TCAinibição de asprodutos,
cycle serve precursors foracetil CoA
a variety e NADH,
of different que provovem
path-
malate, ADP, ATP, and phos- aways
fosforilação de PDC,
present in different inativando-a
cell types (Fig. 20.17). This is particularly important in the
e (as well as many other com- central metabolic role of the liver. The TCA cycle in the liver is often called an “open
VII. Intermediários
specific transporters in the
do CAT e reações anapleróticas
cycle” because there is such a high efflux of intermediates. After a high carbohydrate
A. Intermediários
ndrial membrane that trans- meal, citratedo CAT
efflux andsão precursores
cleavage to acetyl CoApara vias
provides biossintéticas:
acetyl units for cytosolic fatty
1. Importante
ds between the mitochondrial no fígado. CAT é chamado de “ciclo
acid synthesis. During fasting, gluconeogenic precursors are aberto” no tofígado
converted malate,pois há
tosol in exchange for a com-
grande
ar charge. In contrast, CoASH,
whichefluxo demitochondria
leaves the intermediários for cytosolic gluconeogenesis. The liver also uses TCA
cycle intermediates to synthesize carbon skeletons of amino acids. Succinyl CoA may
a) Depois
her CoA derivatives, NAD" and de refeição, efluxo de citrato e sua transformação em acetil CoA
be removed from the TCA cycle to form heme in cells of the liver and bone marrow.
oxaloacetate, are not trans-
fornece
In the brain,acetil
etabolically significant rate. To
para ais síntese
!-ketoglutarate deglutamate
converted to ácidos and graxos
then to #-aminobutyric acid
lic acetyl CoA, many cellsb) Durante jejum, precursores da neoglicogênese
(GABA), a neurotransmitter. In skeletal muscle, !-ketoglutarate is são convertidos
converted to gluta- a malato
que
te to the cytosol, where it ismine,sai daismitocôndria
which transported through para sofrer
the blood neoglicogênese
to other tissues.
etyl CoA and oxaloacetate by
c) Succinil CoA pode gerar o grupo heme em células do fígado e da medula
B. Anaplerotic Reactions
óssea
Removal
d) α- of any of the intermediates
cetoglutarato pode serfrom the TCA cycle
convertido removes the 4 carbons
a glutamato, that
que é transformado
are used to regenerate oxaloacetate during each turn of the cycle. With depletion of
em ácido it-isaminobutírico
oxaloacetate, (GABA
impossible to continue - neurotransmissor)
oxidizing acetyl CoA. To enable the TCA

Acetyl CoA

Amino acid Fatty acid

synthesis Oxaloacetate Citrate synthesis

TCA
cycle
Gluconeogenesis Malate
Amino acid
α –Ketoglutarate synthesis

Succinyl CoA
Neurotransmitter
(brain)

Heme
synthesis

B. ReaçõesFig. 20.17. Efflux of intermediates from the TCA cycle. In the liver, TCA cycle intermedi-
anapleróticas:
ates are continuously withdrawn into the pathways of fatty acid synthesis, amino acid syn-
1. A remoção dos intermediários
thesis, gluconeogenesis, do CAT
and heme synthesis. impede
In brain, a regeneração
!-ketoglutarate is converted tode oxaloacetato e,
gluta-
portanto, fica impossível continuar o cíclo. Para que isso não ocorra, a célula
mate and GABA, both neurotransmitters.

tem diversas vias para repor esses intermediários, chamadas de reações

anapleróticas.
2. Piruvato carboxilase:
a) É uma das enzimas anapleróticas mais importantes
CHAPTER 20 / TRICARBOXYLIC ACID CYCLE 375
b) Catalisa a reação que adiciona CO2 a piruvato para formar oxalacetato
ls have to supply enough four-carbon intermediates from COOH
ate or certain amino acids to compensate for the rate of ATP +
–
HCO3 +C O
ctions that replenish the intermediates of the TCA cycle
CH3
tic (“filling up”).
Pyruvate

pyruvate biotin
XYLASE IS A MAJOR ANAPLEROTIC carboxylase + Acetyl CoA

COOH
ne of the major anaplerotic enzymes in the cell. It cat-
to pyruvate to form oxaloacetate (Fig. 20.18). Like most C O + ADP + Pi
rboxylase contains biotin, which forms a covalent inter- CH2
action requiring ATP and Mg2! (see Fig. 8.12, Chap. 8). COO–
n transferred to pyruvate to form the carboxyl group of
Oxaloacetate
s found in many tissues, such as liver, brain, adipocytes, Fig. 20.18. Pyruvate carboxylase reaction.
function is anaplerotic. Its concentration is high in liver Pyruvate carboxylase adds a carboxyl group
there is a continuous removal of oxaloacetate and malate from bicarbonate (which is in equilibrium with
ter the gluconeogenic pathway. CO2) to pyruvate to form oxaloacetate. Biotin
s activated by acetyl CoA and inhibited by high concen- is used to activate and transfer the CO2. The
 c) É encontrada em tecidos como o nervoso, hepático, adiposo e em
fibroblastos. Sua concentração é mais alta no fígado e no rim, onde
intermediários do CAT são removidos para a neoglicogênese
d) É ativada por acetil CoA
3. Degradação de aminoácidos:
a) A oxidação de diversos aminoácidos converte seu esqueleto carbônico em
intermediários de 4 ou 5 carbonos do CAT, que pode, daí, regenerar
oxaloacetato
(1) Alanina e serina entram pela piruvato carboxilase
(2) Em todos os tecidos com mitocôndrias com excessão do fígado, a
oxadação de isoleucina e valina a succinil CoA é uma importante via
anaplerótica
(3) Glutamina é retirada do sangue, convertida a glutamato, que é
376
transformado em α- cetoglutarato
SECTION FOUR / FUEL OXIDATION AND THE GENERATION OF ATP

Amino Pyruvate
acids Carbohydrates
CO2 Fatty acids
ATP Amino acids
1
ADP + Pi
Acetyl CoA

Oxaloacetate
Citrate
Aspartate
5

Malate Isocitrate

Amino
CO2 acids

4 TA
Amino
Fumarate α – Ketoglutarate Glutamate
acids
2
CO2 GDH
Succinate Succinyl CoA NADH + NAD+
NH4
3

Valine
Isoleucine Propionyl CoA Odd chain fatty acids

Fig. 20.19. Major anaplerotic pathways of the TCA cycle. 1 and 3 (blue arrows) are the two
major anabolic pathways. (1) Pyruvate carboxylase (2) Glutamate is reversibly converted to
!-ketoglutarate by transaminases (TA) and glutamate dehydrogenase (GDH) in many tissues.
(3) The carbon skeletons of valine and isoleucine, a 3-carbon unit from odd chain fatty acid
oxidation, and a number of other compounds enter the TCA cycle at the level of succinyl
CoA. Other amino acids are also degraded to fumarate (4) and oxaloacetate (5), principally
in the liver.

CLINICAL COMMENTS

Otto Shape. Otto Shape is experiencing the benefits of physical condi-

In skeletal muscle and other tis-
sues, ATP is generated by anaero-
bic glycolysis when the rate of aer-
obic respiration is inadequate to meet the
rate of ATP utilization. Under these circum-
stances, the rate of pyruvate production
exceeds the cell’s capacity to oxidize NADH
in the electron transport chain, and hence, to
oxidize pyruvate in the TCA cycle. The
excess pyruvate is reduced to lactate.
Because lactate is an acid, its accumulation
affects the muscle and causes pain and
swelling.
tioning. A variety of functional adaptations in the heart, lungs, vascular
system, and skeletal muscle occur in response to regular graded exercise.
The pumping efficiency of the heart increases, allowing a greater cardiac output
with fewer beats per minute and at a lower rate of oxygen utilization. The lungs
extract a greater percentage of oxygen from the inspired air, allowing fewer respi-
rations per unit of activity. The vasodilatory capacity of the arterial beds in skeletal
muscle increases, promoting greater delivery of oxygen and fuels to exercising mus-
cle. Concurrently, the venous drainage capacity in muscle is enhanced, ensuring that
lactic acid will not accumulate in contracting tissues. These adaptive changes in
physiological responses are accompanied by increases in the number, size, and
activity of skeletal muscle mitochondria, along with the content of TCA cycle
enzymes and components of the electron transport chain. These changes markedly
enhance the oxidative capacity of exercising muscle.

Ann O’Rexia. Ann O’Rexia is experiencing fatigue for a number of

reasons. She has iron deficiency anemia, which affects both iron-
containing hemoglobin in her red blood cells, iron in aconitase and
succinic dehydrogenase, as well as iron in the heme proteins of the electron