Exam 2 Pyruvate Dehydrogenase and the Citric Acid Cycle Flashcards
what are the goals of the PDH reaction?
- oxidize pyruvate (release CO2)
- collect e- from this oxidation as NADH
- activate remaining acetyl group as a “high energy” thioester (acetate to acetyl-CoA)
what problem does PDH effectively deal with?
the alpha-decarboxylation of an alpha-keto acid which forms a highly unstable carbanion
what are the basics of the PDH structure?
3 different active sites, 5 types of subunits
how many E1 subunits are there?
240
dihydrolipoyl transacetylase
the E2 subunits and in some species E3 binding proteins of the PDH complex
how many dihydrolipoyl transacetylase subunits are there in PDH?
60
of the 60 dihydrolipoyl transacetylase subunits in PDH, which categories do the subunits fall into for MAMMALS?
48 E2s
12 E3 BPS
of the 60 dihydrolipoyl transacetylase subunits in PDH, which categories do the subunits fall into for GRAM POSITIVE?
all 60 are E2 subunits
dihydrolipoyl dehydrogenase
E3 subunits of PDH
how many dihydrolipoyl dehydrogenase subunits are in the PDH complex?
24
what is the structure term of the PDH complex?
dual-layered pentagonal dodecahedron
why is the PDH a dual-layered pentagonal dodecahedron?
core of E2 subunits and E3 BPs with an outer shell of E1s and E3s
what are the 5 cofactors required for PDH?
thiamin pyrophosphate (TPP), lipoate/lipoamide, coenzyme A, FAD, NAD+
TPP PDH cofactor
often used for alpha-decarboxylation of alpha-keto acids; provides an electron sink for an unstable electron pair
how is TPP bound to PDH?
E1 prosthetic group
lipoate/lipoamide PDH cofactor
derivatized to enzymes that use it via Lys side chain
how is lipoate/lipoamide bound to PDH?
E2 prosthetic group
Coenzyme A PDH cofactor
provides the thiol for our “high energy” thioester
how is coenzyme A bound to PDH?
transiently associated with enzymes that use it; used by E2 subunits (dihydrolipoyl transacetylase”)
FAD PDH cofactor
oxidation/reduction reactions; many reactions between FAD (fully oxidized) and FADH2 (fully reduced)
how is FAD bound to PDH?
E3 subunit prosthetic group
NAD+ PDH cofactor
oxidation/reduction reactions; “pyridine nucleotide” cofactors
how is NAD+ bound to PDH?
transiently associated with enzymes that use it
what is the mechanism stage for E1 subunits?
decarboxylation of pyruvate and acetylation of lipoamide
what is the mechanism stage for E2 subunits?
acetyl transfer from lipoamide to CoA-SH
what is the mechanism stage for E3 subunits?
oxidation of reduced lipoamide and reduction of NAD+ to NADH
what is the first step/reaction of the CAC?
oxaloacetate + acetyl-CoA –> citrate (achiral)
what is the first step/reaction of the CAC catalyzed by?
citrate synthase
what is the delta G of the citrate synthase reaction?
-31.4 kJ/mol
what is the intermediate of the citrate synthase reaction?
S-citryl-CoA
what is the second step/reaction of the CAC?
citrate <–> 2R, 3S isocitrate
what is the second step/reaction of the CAC catalyzed by?
aconitase
what is the intermediate of the aconitase reaction?
cis-aconitase
what is the delta G of the aconitase reaction?
approx. 0
what does the aconitase rxn utilize?
4Fe/4S cluster, Arg580, Asp/His diade, Ser642 residue
what is the third step/reaction of the CAC?
2R, 3S isocitrate –> alpha-ketoglutarate
what is the third step/reaction of the CAC catalyzed by?
isocitrate dehydrogenase
what is the intermediate of the isocitrate dehydrogenase reaction?
oxalosuccinate
what does the third step/reaction of the CAC signify?
1st release of CO2 and capture of NADH2 in the CAC
what is the delta G of the isocitrate dehydrogenase reaction?
approx. -20 kJ/mol
what is the fourth step of the CAC?
alpha-ketoglutarate + HS-CoS + NAD+ –> succinyl-CoA + NADH + CO2
what is the fourth step of the CAC catalyzed by?
alpha-ketoglutarate dehydrogenase
what is special/cool about alpha-ketoglutarate DH?
it’s mechanism is identical to PDH: same multiplicity of subunits, same 5 cofactors (TPP, lipoamide, CoA-SH, FAD, NAD+)
what is different about alpha-ketoglutarate DH compared to PDH?
the subunits are differently named
transsuccinylase vs. transacetylase
what occurs during the alpha-ketoglutarate DH reaction?
the 2nd NADH is generated and the 2nd CO2 is released (not from our initial acetyl group either)
what is the delta G of the alpha-ketoglutarate DH reaction?
-40kJ/mol
what is the fifth step of the CAC?
succinyl-CoA + Pi + GDP –> succinate + HS-CoA + GTP
what is the fifth step of the CAC catalyzed by?
succinyl-CoA synthetase
what is the delta G of the succinyl-CoA synthetase?
approx. 0
after part 5 of the CAC (succinyl-CoA synthetase reaction), what has occurred?
2 carbons released as CO2, 2 electron pairs captured as NADH, 1 GTP (ATP) synthesized
what is the sixth step of the CAC?
succinate + FAD –> fumurate (trans) + FADH2
what is the sixth step of the CAC catalyzed by?
succinate dehydrogenase
how is FAD bound to succinate dehydrogenase?
prosthetic group bound to SDH (FAD/FADH2 can’t leave the enzyme)
what is the only membrane-bound enzyme of the CAC?
succinate dehydrogenase
where is succinate dehydrogenase bound?
associated with the inner mitochondrial membrane
what is succinate dehydrogenase also known as?
Complex II (a complex of respiratory electron transport system)
what is the seventh step of the CAC?
fumarate <—> L-malate (water into reaction)
what is the seventh step of the CAC catalyzed by?
fumarase
what is the delta g of the fumarase reaction?
approx. 0
what is noteworthy about fumarase?
it operates near catalytic perfection
what is the eighth step of the CAC?
L-malate + NAD+ —> oxaloacetate +NADH + H+
oxaloacetate + NADH + H+ —-> L-malate + NAD+
what is the eighth step of the CAC catalyzed by?
malate dehydrogenase
what occurs during the malate dehydrogenase reaction of the CAC?
the 4th electron pair is collected for RET (3rd NADH)
what is the delta G of the malate dehydrogenase reaction?
+29.7 kJ/mol
what upregulates PDH and CAC?
relatively low energy charge… AKA
high [ADP] and [AMP]
low [ATP]
high [NAD+/NADH]
what downregulates PDH and CAC?
relatively high energy charge… AKA
low [ATP] and [AMP]
high [ATP]
low [NAD+]/[NADH]
Pyruvate —> acetyl-CoA
thermodynamically irreversible, metabolically irreversible in mammals
after pyruvate is converted to acetyl-CoA, what are the options of C?
carbon is committed to either:
- generation of ATP via oxidative phosphorylation (oxidation via the CAC)
- synthesis of fatty acids and other lipids
what are the three inhibitors of PDH?
NADH, acetyl-CoA, ATP
how does NADH inhibit PDH?
inhibits E3 subunits; outcompetes NAD+ in E3 active sites, so it can’t reoxidize lipoamide
how does acetyl-CoA inhibit PDH?
inhibits E2 subunits; competes with CoA-SH… high acetyl-CoA will turn off process that generates a lot of acetyl-CoA
how does ATP inhibit PDH?
allosterically inhibits E1 subunits; inhibition is enhanced by fatty acyl-CoA’s; marker for the fasting state (keeps PDH inactive)
what can and cannot be used for gluconeogenesis??!!
acetyl-CoA cannot be used for gluconeogenesis; pyruvate can
what is an activator of PDH?
AMP
how does AMP activate PDH?
allosterically activates E1 subunits
Can PDH be phosphorylated/dephosphorylated in mammals?
yes
what subunits of PDH can be phosphorylated?
E1alpha subunits
what are the three phosphorylatable sites on the E1alpha PDH subunits?
Ser246, Ser271, Ser 203
what occurs when any of the three phosphorylatable E1alpha PDH subunit sites are phosphorylated?
inactivation of PDH subunits
phosphorylated PDH
inactive
dephosphorylated PDH
active
what enzyme phosphorylates PDH?
pyruvate dehydrogenase kinase (PDK)
what are the four isozymes of PDK?
PDK1, PDK2, PDK3, PDK4
what are all isozymes of PDK activated by?
acetyl-CoA and NADH
what are all isozymes of PDK inactivated by?
pyruvate
where is PDK1 found?
pancreatic Beta cells and cardiomyocytes
what is the target site of PDK1?
site 3 (Ser203)
where is PDK2 found?
cardiomyocytes and hepatocytes
what is the target site of PDK2?
site 1 (Ser246)
PDK3
weak expression in most tissues
where is PDK4 found?
cardiomyocytes, skeletal myocytes, hepatocytes, adipocytes
what is the target site of PDK4?
site 2 (Ser271)
what is PDK4 used/seen as?
long-term regulatory mechanism of PDH
what increases PDK4 expression?
increase stress… increase fasting state —> produces glucocorticoids which increases expression of PDK4
glucocorticoids
major regulator of expression (ex: cortisol)
what decreases PDK4 expression?
insulin (increase/presence)
what enzyme dephosphorylates PDH?
pyruvate dehydrogenase phosphatase (PDP)
what are the two isozymes of PDP?
PDP1 and PDP2
where is PDP1 found?
skeletal muscle
what is PDP1 activated by?
Ca2+ (direct response to muscle contraction)
where is PDP2 found?
hepatocytes, adipocytes, cardiomyocytes
what activates PDP2?
insulin (due at least in part to the activity of protein kinase B)
T or F: PDP2 is at the level of expression
true
what are PDP1 and PDP2 both activated by?
Mg2+ (connection to energy charge)
when is Mg2+ sequestered and unavailable to activate PDP1 or PDP2?
high [ATP]
low [ADP] and [AMP]
when is Mg2+ released and available to activate PDP1 and PDP2?
low [ATP]
high [ADP] and [AMP]
what are the three activators of isocitrate dehydrogenase?
ADP, NAD+, Ca2+
what are the two inhibitors of isocitrate dehydrogenase?
NADH and ATP
what are the two inhibitors of alpha-ketoglutarate DH?
NADH (E3s), succinyl-CoA (E2s), and ATP (allosteric control of E1s)
what activates alpha-ketoglutarate DH?
AMP (allosteric control of E1s)
what does regulation of alpha-ketoglutarate DH do?
preserves S-C structure for Glu, Gln, Arg, Pro biosynthesis
what can glutamine (Glu) be used for?
biosynthesis of pyrimidine and purine
