Lehninger Ch. 16
BIO 322 Recitation 3 / Spring 2013
•Glycolysis – complete oxidation of glucose
•Respiration - pyruvate to water and carbondioxide
•Cellular Respiration occurs in three major stages.
1) Glucose,FA, AA – oxidized to yield 2C fragments in
the acetyl group of acetyl-CoA.
2) Acetyl groups are fed in to TCA cycle, enzymatically
oxidizes them to CO2. Energy conserved as NADH and
FADH2.
3) NADH and FADH2 are oxidized to give up protons
(H+) and electrons. Electrons are transfered to O2 via
ETC. (Release of large amount of ATP via oxidative
phosphorylation)
•Pyruvate Dehydrogenase Complex
•5 cofactors, 4 of them are vitamins
•Covalent modification and allosteric
regulation example
•Similarity in structure, cofactor req., rxn
mech. to
Oxidative decarboxylation – irreversible
oxidation process, carboxyl group from
pyruvate is removed from as CO2.
NADH to ETC (Hydride ion – 2
electrons to oxygen) – 2,5 ATP per
NADH
•α-ketoglutarate dehydrogenase of TCA
cycle
•Branched chain α-keto acid
dehydrogenase, from oxidative pathway
of several AA.
• TPP (Thiamine pyrophophate) –
thiamine (B1)
• FAD (Flavin adenine
dinucleotide) – riboflavin (B2)
• Coenzyme A (CoA, CoA-SH) –
pantothenate (B5)
• NAD (Nicotinamide adenine
dinucleotide) – niacin (B3)
• Lipoate
• CoA – thiol (-SH) group – acyl carrier
• Acyl forms covalent linkage to thiol group
(High standard free energy of hydrolysis) –
transfer potential
•Lipoate – two thiol groups – reversible oxidation to
disulphide linkage
• Can undergo redox rxns – electron and acyl
carrier.
1) Identical rxn to pyruvate decarboxylase (Pyruvate to oxalaacetate), C1 of pyruvate to
CO2. C2 of pyruvate in the aldehyde form to TPP as hydroxyethyl group. Rate limiting
and Substrate Specificity.
2) Hydroxyethyl group into carboxylic acid (acetate) form. 2 electrons removed in this
reaction reduce –S-S of lipoyl group of E2 to two thiol (-SH) groups.
3) Acetyl group from above redox, esterified first to one lipoyl-SH group, then transesterified
to CoA to form acetyl-CoA.
4) Regeneration oxidized of disulphide form of lipoyl group in E2.
2e + acetyl from pyruvate in E1 to lipollysyl arms of E2, transferring them to E3.
Substrate chaneling – intermediates never leave the complex, substrate of E2 is kept at high
conc., protects acetyl group to be used by other enzymes.
Thiamine deficiency – Beriberi – neuronal dysfunction (white rice, large amounts of alcohol
Consumption, high pyruvate concentration in the blood.
•Citrosysl CoA – transient intermediate
•Free CoA and citrate as products
•The large negative free energy is essential to
the operation TCA cycle because the
concentration of oxaloacetate is normally
very low.
•CoA sent back to PDH.
• Reversible – cis-aconitate intermediate
• Addition of water
•At pH 7.4, 25˚C the eq. mix contains less 10%
isocitrate.
•However in the cell, because isocitrate is
rapidly consumed in the next step of the TCA
cycle, isocitrate formation is favored.
•Contains iron-sulphur center (iron is used for
heme, cytochromes, Fe-S proteins)
•Transferrin – iron transporter
•Ferritin – iron storage
•When cells are depleted of iron, acotinase
loses its activity , apo-acotinase binds specific
mRNAs for transferrin receptor and ferritin.
 Regulating protein synthesis at translational
level.
• Oxidative decarboxylation
•NAD+ and NADP+ requiring enzymes
• Identical Rxn to PDH
• E1 is structurally similar but AA sequences differ, thus
they have different binding specifity.
PDH – pyruvate
α-ketoglutarate dehydrogenase – α-ketoglutarate
E2 – similar both have lipoyl moieties.
E3 – identical
CoA as the carrier of succinyl group
• Enzyme phosphorylated at a His residue in the
active site.
• This phosphoryl group is transferred to ADP or
GDP, dependent on the type of isozymes.
• Stabilized active site (phosphoenzyme
intermediate) by two subunits oriented in such a way
that they confer a partial positive charge on the
negatively charged phospho-His residue.
•Formation of ATP at the expense of energy released
by oxidative carboxylation is substrate level
phosphorylation
Mitoch. inner memb.
3 FE-S and FAD – electrons pass
before these ETC.
1,5 ATP molecules per electrons
(respiration linked phosphorylation)
• Because oxaloacetate is removed by step 1,
the concentration of oxaloacetate is kept
very low.
•This condition pulls malate dehydrogenase
rxn to the formation of oxaloacetate.
• Supply NADH and FADH2 to ETC for
oxidative phosphorylation
•2 e from NADH to oxygen – 2,5 ATP
•2 e from FADH2 to oxygen – 1,5 ATP
•32 ATP per glucose molecule
•32 x 30,5 kj/mol = 976 kj/mol
34% of theoretical max
When corrected for actual free
energies, the process is 65% efficient.
• Some modern microorganisms lack
α-ketoglutarate dehydrogenase
•They have reversible enzymes to
carry out from oxaloacetate to
succinyl-CoA ( reverse of the cycle)
• Fermentation because NADH from
isocitrate oxidation is recycled to
NAD+ by reduction of oxaloacetate to
succinate.
Removed intermediates for
biosyn are replenished by
anaplerotic rxns.
Allosteric inhibiton of PDH by long chains fatty acids, ATP/ADP,
NADH/NAD, acetyl-CoA/CoA
Allosteric activation of PDH by AMP, CoA, NAD+
Covalent protein modification – PDH inhibited by phosphorylation of E1
at Ser residue on one of the subunits. This kinase is allosterically
regulated by ATP levels.
Flow of metabolites in TCA cycle 3 Factors:
1) Substrate Availability
2) Inhibition by accumulating products
3) Allosteric feedback inhibition that catalyze early
steps of the cycle