phosphogluconate pathway, PDH, TCA Flashcards
1
Q
what are the 2 phases of the phosphogluconate pathway
A
oxidative and non-oxidative
2
Q
what is the purpose of the PPP
A
- reduction of NADP+ to NADPH for reductive biosynthetic rxns or to counter oxygen radicals
- Synthesis of ribose 5 phosphate (precursor for nucleotides, RNA,DNA, and coenzymes ATP, NADH, FADH2, coenzyme A )
3
Q
NADPH is used for the synthesis of what
A
fatty acids and other molecules
4
Q
what tissues have little shunt actvity
A
Tissues that produce little fat and are not actively dividing have little shunt activity (i.e. muscle)
5
Q
what is used for nucleic acid synthesis
A
Ribose phosphate are used for nucleic acid synthesis
6
Q
what happens to tissues only need NADPH and not any other product (such as ribose 5 phosphate)
A
In tissues requiring NADPH, ribulose-5-P is recycled to G-6-P
7
Q
what is the general rxn that happens in the non-oxidative phase
A
six 5-carbon molecules are converted to 5
6-carbon molecules, thus regenerating glucose-6-phosphate
8
Q
what is the first rxn of PPP
A
G-6-P + NADP+ to 6-phospho-δ-gluconolactone + NADPH
G-6-P dehydrogenase
NADP+ is reduced to NADPH
9
Q
what kind of rxn happens in the 1st PPP rxn
A
redox: G6P oxidized and NADP+ is reduced
10
Q
what is the 2nd rxn of PPP
A
6-phospho-δ-gluconolactone reacts with water to form 6-phosphogluconate (hydrolysis)
enzyme: lactonase
cofactor: Mg2+
11
Q
3rd rxn in PPP
A
6-phosphogluconate + NADP+ reacts to form D-Ribulose-5-phosphate + NADPH + H+ + CO2
enzyme: 6-phosphogluconate dehydrogenase
Oxidation and decarboxylation rxn
12
Q
4th rxn in PPP
A
D-Ribulose-5-phosphate to D-ribose 5 phosphate
phosphopentose isomerase
13
Q
what creates superoxide radicals (*O2)
A
mitochondrial respiration, ionizing radiation, sulfa drugs, herbicides, antimalarials, divicine
14
Q
what do superoxide radical *O2 become and what do they react with to become that.
A
they become H2O2; they react with 2 H+ and an e-
15
Q
what does H2O2 become if not reduced
A
a hydroxyl free radical *OH
16
Q
a hydroxyl free radical *OH cause what type of damage? What do they damage?
A
oxidative damage to lipids, proteins, DNA
17
Q
what cells keep high levels of NADPH/NADP+ ratios to combat oxidative damage?
A
cells of lens and cornea; erythrocytes
18
Q
how does NADPH help fight oxidative damage
A
it reduces GSSG to 2 GSH (glutathione)
enzyme: glutathione reductase
19
Q
what does 2GSH do?
A
it reacts with H2O2 to form 2 H2O
it can (also) inhibit reactive hydroxyl radical damage to tissues
20
Q
R-5-P recycling involves what
A
• R-5-P recycling involves a complex series of reactions involving transketolase and transaldolase
21
Q
what intermediates are formed in R-5-P recycling
A
• 7-carbon, 4-carbon, and 3-carbon sugar phosphates are intermediates
22
Q
what does epimerase do
A
converts between ribose 5 phoshate and xylulose 5 phosphate
23
Q
what does transketolase do to R5P and X5P
A
makes sedoheptulose 7 phosphate and G3P
24
Q
what does transaldolase do to sedoheptulose 7 phosphate and G3P
A
makes F6P and Erythrose 4 phoshate
25
what is F6P converted to
G6P
26
what is Erythrose 4 phoshate and X5P (from another origin) converted to and by what enzyme
F6P and G3P by transketolase
27
what can happen to G3P made from X5P
enter gluconeogenesis
Or react with sedoheptulose 7 phosphate to make F6P and erythrose 4 Phosphate via transaldolase rxn
28
what is made in ribose 5 phosphate recycling (generally)
six 5 carbon sugars make five 6 carbon sugars
29
what is a disease of the PPP: describe it
Wernicke-Korsakoff Syndrome:
-caused by TPP deficiency
```
-exarcerbated by mutation in the
transketolase gene (lowered affinity for
TPP); more sensitive to thiamine deficiency
```
-slows down PPP
30
TTP is a cofactor what what in PPP
transketolase
31
what is the general names for the 2 substrates of transketolase
aldose acceptor and ketose donor
32
what are the 2 fates of G6P and what influences its fate
• G-6-P is partitioned between glycolysis and the PPP, depending on current needs of cell and on conc. of NADP+ in cytosol
33
what inhibits and what activates G6P entrance into PPP
-NADP+ is allosteric activator and NADPH is allosteric inhibitor
34
what is pyruvate oxidized to in respiration
Pyruvate produced by glycolysis is further oxidized
| to H20 and C02 in an aerobic phase of catabolism
35
what are the 3 major stages of cellular respiration
1) Organic fuel molecules - glucose, fatty acids, some amino acids are oxidized to yield 2-carbon fragments in the form of acetyl group of acetyl-CoA
2) The acetyl groups enter the citric acid cycle, which oxidizes them to C02; energy released is conserved in reduced electron carriers NADH and FADH2
3) Reduced coenzymes are themselves oxidized, giving up H+ and electrons; electrons are transferred to 02- via the mitochondrial electron transport chain
During electron transfer, the large amount of energy
released is conserved in the form of ATP
36
what is acetyl CoA made from
Oxidation of fatty acids, glucose, and some amino acids yield acetyl-CoA
37
what transports pyruvate into mitochondria
pyruvate translocase (cotransport with H+)
38
what is the delta G of the PDH
-33 kj/mol
39
what are the cofactors of PDH
TPP, lipoate (lipoic acid), FAD+, NAD+, CoA-SH
40
what are the subtrates and products of PDH
pyruvate to acetyl CoA , Co2 and NADH
41
what is the rxn of PDH called
oxidative decarboxylation: irreversible
42
what happens to NADH formed
donate hydride to ETC
43
how many ATP's per NADH and FADH2
2.5; 1.5
44
what happens in the first part of PDH
pruvate is releases CO2 and forms Hydroxyethyl TPP (HETPP)
enzyme: pyruvate dehydrogenase
45
what happens in 2nd part of PDH
Hydroxyethyl group is transfered to lipoic acid and is oxidized to form acetyl dihydrolipoamide
enzyme: dihydrolipoyl transacetylase
46
what happen in 3rd part of PDH
acetyl group is transfered to CoA
enzyme: dihydrolipoyl transacetylase
47
4th part of PDH
Dihydrolipoamaide is reoxidized and lipoic acid is regenerated
enzyme: dihydrolipoyl dehydrogenase
48
what does FAD stand for
Flavin adenine dinucleotide
49
what does TPP do?
Thiamine Pyrophospate(TPP): involved incleavage of bonds adjacent to carbonyl groups and transfer of activated acetaldehyde groups from one C to another
decarboxylates pyruvate
50
what does lipoic acid do
serves as both an electron carrier and an acyl carrier
51
what does TPP do
1) TPP reacts with pyruvate, which undergoes decarboxylation.
C-1 of pyruvate is released as CO2; C-2 of pyruvate, now an
aldehyde, is attached to TPP
52
what is the ring in TPP called? what is it involved in?
thiazolium ring: C2 of ring attaches with what was C2 of pyruvate (active acetaldehyde). C1 of pyruvate was released as CO2
53
what does lipoic acid do?
-contains 2 thiol groups which are found in either reduced (dithiol) or oxidized (disulfide) forms;
functions as an electron carrier or an acyl carrier
-Pyruvate dehydrogenase transfers two electrons and the acetyl
group to lipoic acid. This reduces the oxidized lipoic acid and transfer
of the acetyl group to the lipoyllysyl group to form an acetyl thioester.
54
what does CoA do
- Functions as an acyl carrier by forming a thiol ester bond
between its thiol group and the acyl group
3) Transter of the acetyl group to CoA-SH, forming acetyl-CoA
4) Reoxidation of lipoic acid, transferring reducing equivalents to NAD+
55
Which are co-substrates in PDH and which are prosthetic groups
CoA-SH and NAD+ are cosubstrates; TPP, lipoate andn FAD are prosthetic groups
56
Wernicke Encephalopathy
Characterized by ocular
abnormalities, ataxia, and state of global
confusion. commonly (but not exclusively)
associated with chronic alcohol abuse.
57
Beriberi:
manifestations include fatigue, irritability,
sleep disturbance, abdominal pain, anorexia,
leading to swelling, pain, paralysis, and death
58
Pyruvate Dehydrogenase Complex Deficiency:
```
manifested in infancy or later childhood
by progressive neural symptoms, including
intermittent ataxia, poor muscle tone,
abnormal eye movements, or seizure.
Patient manifests elevated blood lactate
```
59
what do wernicke encephalopathy, beriberi and PDH deficiency have in common?
thiamine deficiency?
60
overview of TPP
1) Thiamine Pyrophospate (TPP): contains thiamine (vitamin B1)
and a pyrophosphate group. Lack of vitamin B1 in the diet
leads to beriberi, a condition characterized by accumulation of
body fluids, pain, paralysis, loss of neural function and death.
The brain usually obtains all its energy from aerobic oxidation
of glucose and therefore sensitive to thiamine deficiency.
Beriberi occurs in populations that consume polished white rice
(lacks hulls in which thiamine is found) and in alcoholics.
61
overview of FAD
2) Flavin Adenine Dinucleotide (FAD) consists of riboflavin (vitamin
B2) and adenine dinucleotide; accepts 2 H atoms (2 electrons
and two protons) to form its reduced form FADH2
62
overview of NAD+
3) NAD+ consists of niacin (a vitamin) and adenine dinucleotide;
accepts a hydride (2 electrons and 1 proton) to form its
reduced form NADH
63
overview of CoA
```
4) Coenzyme A (CoA-SH) consists of a phosphoadenine dinucleotide,
pantothenic acid (a vitamin) and β-mercaptoethylamine;
functions as acyl carrier by forming thiol ester bond
```
64
lipoic acid overview
5) Lipoic Acid: contains 2 thiol groups which are found in either
reduced (dithiol) or oxidized (disulfide) forms; functions as an
electron carrier or an acyl carrier
65
about E1 of PDH
1) Pyruvate dehydrogenase, E1
PDC has 12 copies of E1, each
with two identical subunits;
contains bound TPP
66
about E2 of PDH
```
2) Dihydrolipoyl transacetylase,E2
core of the PDC has 60 copies
of E2; lipoate is covalently
attached to lysine groups in E2
lipollysyl groups provide long
flexible arms to deliver attached
acyl groups from one active site to
another
```
67
about E3 of PDH
3) Dihydrolipoyl dehydrogenase,E3
PDC has 6 copies of E3, each
with two identical subunits;
contains bound FAD
68
generally what are the rxn steps of PDH and what enzyme catalyzes each step
Pyruvate dehydrogenase, E1:
1) Oxidative decarboxylation of pyruvate occurs after reaction with TPP; C-1
of pyruvate released as CO2; C-2, now an aldehyde, is attached to TPP
Dihydrolipoyl transacetylase, E2:
2) Hydroxyethyl group is oxidized to carboxylic acid (acetate); 2 e- now
reduce disulfide bonds in lipoyl group on E2 to 2 thiol groups; acetate is
transferred to one of thiol groups on E2
3) Acetate is transesterified to CoA to yield acetyl-CoA
Dihydrolipoyl dehydrogenase, E3:
4) Thiol moieties of lipoate are oxidized to regenerate the disulfide bond;
2 H atoms reduce FAD (associated with E3); Hydride ion is transferred
from FADH2 to NAD+ to form NADH
69
how is PDH regulated
PDC is regulated both allosterically and by chemical
modification:
Inhibited by: ATP, acetyl-CoA, NADH, fatty acids,
phosphorylation of serine on E1
Activated by: AMP, CoA-SH, NAD+, Ca2+ (signal for
muscle contraction and increased demand for ATP)
*FIND better diagram*
70
what are 4 general features of TCA
1) Four steps that generate NADH or
FADH2
2) One step that generates ATP/GTP
3) Oxidation dependent on availability of
oxaloacetate
```
4) Entry of 2 carbonfrom acetyl-CoA
…two mol.of C02 leave
-one mol of OAA used
…one mol. of OAA is regenerated,
Thus no net change
```
71
What is the purpose of having
| a circular pathway?
1) Citric acid cycle intermediates are
starting materials in anabolic pathways,
and they are endproducts of catabolic pathways such as degradation of glucogenic amino
acids
2) the oxidation of acetyl units would be coordinated with other pathways such as the synthesis of
glucose
72
step 1 of TCA
Acetyl CoA + OAA + H20--> citrate + CoA-SH
enzyme: citrate synthase
73
describe step 1 of TCA
-what is delta G
- Citrate is formed by the condensation of acetyl-CoA with oxaloacetate; CoA is released
- Irreversible reaction - large negative free energy of reaction (-32,2 kj/mol) is necessary to drive reaction because oxaloacetate is present in very low concentration in cells
74
structure of citrate synthase
```
Structure of citrate
synthase:
Subunits undergo
conformational change
from open (a) to closed
(b) form on binding
oxaloacetate creating
a binding site
for acetyl-CoA
```
75
what makes citrate rxn highly exergonic
```
Hydrolysis of
high energy
thioester
intermediate
(citroyl-CoA)
makes forward
rxn highly
exergonic
```
76
what is the only step in TCA cycle that involves formation of C-C bond
citrate synthase rxn
77
what is the intermediate in citrate synthase intermediate
citryl -CoA
78
conformational change of citrate synthase
Subunits undergo conformational change from open to
closed form on binding oxaloacetate creating a binding site
for acetyl-CoA; forms transient citroyl-CoA that rapidly
undergoes hydrolysis to CoA and citrate
79
step 2 of TCA
citrate --> cis-aconitate --> isocitrate
aconitase in both reactions
80
what is delta G of step 2 of TCA
13.3 kj/mol reversible
81
what types of rxn happen in each step in aconitase rxns
Isocitrate is formed by 2-step mechanism involving dehydration
and hydration via the intermediate cis-aconitate
82
what kind of center does aconitase have? what attaches to it?
iron sulfur center
3 Cys residues of enzyme bind 3 Fe atoms; 4th Fe is bound to
one of the carboxyl groups of citrate and interacts noncovalently
with a hydroxyl group of citrate
83
step 3 of TCA
isocitrate + NAD+ --> oxalosuccinate + NADH and H+--> CO2 + intermediate --> alpha ketogluterate
Oxidation of Isocitrate
(Isocitrate dehydrogenase)
84
what are byproducts of isocitrate dehydrogenase rxn
CO2 and NADH
85
isocitrate dehydrogenase rxn is the first in what 2 categories in the TCA
first oxidation, generating NADH
first rxn where carbon unit is lost
86
step 4 of TCA
Oxidation of a-ketoglutarate + CoA-SH and NAD+ to succinyl-CoA and CO2 with generation of NADH
enzyme: alpha ketogluterate dehydrogenase complex
87
delta G of step 4 of TCA
-33.5; irreversible
88
compare alpha ketogluterate dehydrogenase complex to PDH
virtually identical rxn to PDH
89
step 5 of TCA
Succinyl-CoA is converted to succinate + CoA-SH with generation of GTP
succinate CoA synthetase
90
delta G of step 5 of TCA
-2.9; reversible
91
what is conserved in Succinyl CoA synthetase rxn?
what do the 2 isozymes yield?
The energy of the thioester bond is conserved by phosphorylation
of GDP or ADP to GTP or ATP; two isozymes, one specific for
GDP and the other for ADP
92
step 6 of TCA
Succinate is oxidized to fumarate with the generation of FADH2 from FAD
succinate dehydrogenase
93
enzymatic activity
in Complex II of ETC
94
what happens to reducing equivalents in succinate dehydrogenase rxn
Reducing equivalents are transferred via FAD and iron-sulfur
| centers to ubiquinone
95
what is delta G knot of step 6 of TCA
0; reversible
96
succinate dehydrogenase inhibited by
malonate (similar structure)
97
step 7 of TCA
fumarate + OH- --> carbanion transition state + H+ (from outside)--> Malate
enzyme: fumarase
98
delta G of step 7 of TCA
-3.8
99
step 8 of TCA
L-Malate + NAD+ --> NADH + Oxaloacetate
malate dehydrogenase
100
delta G of step 8 of TCA
29.7 reversible
101
malate dehydrogenase rxn has high positive delta G: how does it proceed
Although this reaction has a high positive G’o, the reaction
proceeds from left to right since oxaloacetate is present at very
low concentrations (<1 μM)
102
what is the net yield of TCA per acetyl CoA
3 NADH, 1 FADH2, 1 GTP (ATP), and
2 CO2 (not same carbons that entered
the cycle as acetyl group)
103
what is the number of total ATPs that result from TCA
10 per acetyl CoA
104
ATP's per glucose molecule
30-32
105
Role of Citric Acid Cycle
| In Anabolism
The TCA cycle provides precursors for other
biosynthetic pathways:
1) a-ketoglutarate is
a precursor of
amino acids and
nucleotides
```
2) Oxaloacetate is
a precursor of
a) amino acids
and can also be
b) converted to
glucose via PEP
```
```
3) Succinyl-CoA is
an intermediate
in synthesis of
porphyrin ring
of heme
```
106
what are 3 fates of PEP
Glucose
AA (serine, glycine, cysteine, phenylalanine, tyrosine, tryptophan)
OAA (via PEP carboxylase)
107
pyruvate can become
acetyl CoA
OAA ( via pyruvate carboxylase (followed by PEP carboxykinase??))
malate ( via malic enzyme)
108
citrate can also become
FAs, sterols
109
alphaketogluterate can also become
glutamate
- glutamate can then become purines or AA's ( glutamine, proline or arginine)
110
succinyl CoA can become
porphyrins, heme
111
OAA can become
aspartate or asparigine
- which can then become pyrimidines
112
what enzyme catlyzes the carboxylation of pyruvate to form OAA? any cofactors?
pyruvate carboxylate
cofactor: biotin
113
what is the role of biotin in carboxylase
Role of biotin:
```
Biotin is attached
to enzyme via
amide bond with
the amino group of
a Lysine, forming a
biotinyl-enzyme
```
```
Biotin acts as a
carrier to
transport the CO2
from one active
site to another on
the same enzyme
```
114
how is TCA cycle regulated
1) Availability of substrates (limits flux)
2) Allosteric regulation at its three exergonic steps.
```
1) Inhibited if:
high ratios of
[ATP/ADP],
[NADH/NAD],
[acetylCoA/CoA]
=energy
sufficient
metabolic state
```
2) Also inhibited
by succinyl-CoA,
citrate, ATP
3) Activated by:
ADP, Ca2+
115
PDH is inhibited/ activated by what
inhibited by ATP, Acetyl CoA, NADH and fatty acids
activated by AMP, CoA, NAD+, Ca2+
116
citrate synthase inhibition/activation
inhibitied by NADH, Succinyl CoA, citrate, ATP
activated by: ADP
117
isocitrate dehydrogenase inhibition/activation
inhibited by : ATP
activated by Ca2+ and ADP
118
alpha ketogluterate dehydrogenase complex regulation
inhibited by: succinyl CoA, NADH
activated by Ca2+
119
what carbon of G6P is released as CO2 in PPP
C1
120
TPP is what
Thiamine pyrophosphate
121
where are all enzymes of TCA located
all except for succinate dehydrogenase are in matrix. Succinate dehydrogenase is in inner mitochondrial membrane
122
malonate is a competitive inhibitor of what
succinate dehydrogenase
123
what are 2 key regulatory enzymes of TCA
what are some compounds they inhibited by
citrate synthase and isocitrate dehydrogenase
ATP and/or NADH
124
what is required for conversion of succinate to fumarate in TCA
FAD
125
For the rxn :
L-Malate + NAD+ ? oxaloacetate + NADH + H+, ?G'° = 29.7 kJ/mol. The reaction as written:
can it occur?
yes, may occur in cells at certain concentrations of substrate and product.
126
The two moles of CO2 produced in the first turn of the citric acid cycle have their origin in the
two carboxyl groups derived from oxaloacetate.
127
what fraction of OAA carbons are used in each cycle of TCA
half
128
The oxidation of 3 mol of glucose by the pentose phosphate pathway may result in the production of
3 mol of pentose, 6 mol of NADPH, and 3 mol of CO2
129
The pentose phosphate pathway represents an oxidation/reduction. What is the most reduced and Oxidized product of this pathway
NADPH and Carbon dioxide respectively.
130
The pentose phosphate pathway involves:
trioses, pentoses, hexoses, heptoses
131
What enzyme converts GTP formed in TCA to ATP?
Nucleotide diphosphate kinase
