Chapter 16 - The citric acid cycle - Biochemistry

Chapter 16 - The citric acid cycle - Biochemistry

1. 16|$The$Citric$Acid$Cycle$
© 2013 W. H. Freeman and Company

2. Only$a$small$amount$of$energy$available$
in$glucose$is$captured$in$glycolysis$
2
ΔG′° = –146 kJ/mol
Glycolysis
Full oxidation (+ 6 O
2
)
ΔG′° = –2,840 kJ/mol
6 CO
2
+ 6 H
2
O
Glucose
Only the 1
st
stage of
Glucose oxidation

3. Cellular$Respira>on$
• "Process"in"which"cells"consume"O
2
"and"produce"CO
2"
• "Provides"more"energy"(ATP)"from"glucose"than"glycolysis"
• "Also"captures"energy"stored"in"lipids"and"amino"acids"
• "Used"by"animals,"plants,"and"many"microorganisms"
""
• "Occurs"in"three"major"stages:""
D "acetyl"CoA"producEon"(from"organic"fuel"molecules)"
D "acetyl"CoA"oxidaEon"(in"the"CAC"to"produce"CO
2
)"
D "electron"transfer"and"oxidaEve"phosphorylaEon"
(reduced"coenzymes"from"CAC"give"their"e
–
’s"to"O
2
"
forming"ATP"in"the"process)"

4. Respira>on:$Stage$1$
AcetylACoA$Produc>on$
• AcEvated"form"of"acetate"
• CDskeleton"of"sugars"and"faKy"
acids"are"converted"to"acetylD
CoA"before"entering"the"CAC"
some%a.a.%enter%CAC%via%other%intermediates%
• Pyruvate$dehydrogenase$
complex$(PDH)$
• MulEple"copies"of"3"enzymes"
• 5"reacEons"by"3"enzymes,"whereby"the"intermediates"remain"bound"
to"the"enzyme"molecule"unEl"forming"the"final"product"
• 5"cofactors"(4"derived"from"vitamins)""

5. Respira>on:$Stage$2$
AcetylACoA$oxida>on$
Generates"
NADH,"FADH
2
,
"
and"one"GTP
"

6. Respira>on:$Stage$3$
Oxida>ve$Phosphoryla>on$
Generates"
a"lot"of"
ATP$
$

7. In$eukaryotes,$citric$acid$cycle$occurs$in$
mitochondria$
• Glycolysis"occurs"in"the"cytoplasm"
• Citric"acid"cycle"occurs"in"the"mitochondrial"
matrix
†"
• OxidaEve"phosphorylaEon"occurs"on"and"in"the"
inner"membrane"
†
Except"succinate"dehydrogenase,"which"is"located"in"the"mitochondrial"
inner"membrane"

8. Conversion$of$Pyruvate$to$AcetylACoA$
• Net"ReacEon:"
– Oxida*ve,decarboxyla*on"of"pyruvate"
– First"carbons"of"glucose"to"be"fully"oxidized""
(remember:"2"pyr/glc)"
• Catalyzed"by"PDH"
– Requires"5"coenzymes"
– TPP,"lipoate,"and"FAD"are"prostheEc"groups"
– NAD
+
"and"CoADSH""are"coDsubstrates"
TPP"–"thiamine"(B1)"
FAD"–""riboflavin"(B2)"
NAD"–"niacin"(B3)"
CoA"–"panthothenic"acid"(B5)"
"
Derived"from:"

9. Structure$of$Coenzyme$A$
• Coenzymes"are"not"a"permanent"part"of"the"enzymes’"structure."
– They"associate,"fulfill"a"funcEon,"and"dissociate"
• The"funcEon"of"CoA"is"to"accept"and"carry"acetyl"groups"""
• Thioesters"have"a"high"acyl"group"transfer"potenEal"(donate"their"
acyl"groups"to"different"groups)"

10. Structure$of$Lipoyllysine$
• ProstheEc"groups"are"strongly"bound"to"the"protein"
– The"lipoic"acid"is"covalently"linked"to"the"enzyme"via"a"lysine"residue"
(lipoyllysine)""
– Undergo"reversible"redox"
reacEons"between"thiols"
and"disulfides;"hence"
can"serve"as"an"electron"
(Hydrogen)"carrier"and"
an"acyl"carrier"

11. Pyruvate$Dehydrogenase$Complex$(PDH)$
• "Large"(up"to"10$MDa)"mulEenzyme"complex""
D "large"enough"to"be"seen"with"cryoEM"
D "pyruvate"dehydrogenase"(E
1)"
D "dihydrolipoyl"transacetylase"(E
2)"
D "dihydrolipoyl"dehydrogenase"(E
3)"
D "each"present"in"mul3ple%copies""
• Advantages"of"mulEenzyme"complexes:""
‒ short"distance"between"catalyEc"sites""
allows"channeling"of""substrates"from"one""
catalyEc"site"to"another"
‒ channeling"minimizes"side"reacEons"
‒ "regulaEon"of"acEvity"of"one"subunit"affects"the"enEre"complex"

12. 3D$Reconstruc>on$from$CryoAEM$data$
The"bovine"enzyme"
Core:"60"idenEcal""
copies"of"E
2
)"
E
1
"acEve"site"has""
bound"TPP"
E
3
"acEve"site"has""
bound"FAD"
Protein"kinase"and"phosphoprotein"phosphatase"are"part"of"the"complex"

13. Overall$Reac>on$of$PDH$
E
1
""
• Step$1:"DecarboxylaEon"of"pyruvate"to"an"aldehyde""
• Step$2:"OxidaEon"of"aldehyde"to"a"carboxylic"acid"
‒ Electrons"reduce"lipoamide"and"form"a"thioester"
E
2"
• "Step$3:""
FormaEon""
of"acetylDCoA""
(product"1)"
E
3
""
• Step$4:""
ReoxidaEon"of"the"lipoamide"cofactor"
• Step$5:""
RegeneraEon"of"the"oxidized"FAD"cofactor"
‒ Forming"NADH"(product"2)"
"

15. The$Citric$Acid$Cycle$(CAC)$
CDC"bond"formaEon"to""
make"citrate""
IsomerizaEon"via""
dehyd./rehydraEon
OxidaEve"decarboxyD"
laEons"to"give"2"NADH"
SubstrateDlevel"phosphorylaEon"to"give"GTP
DehydrogenaEon""
to"give""
reduced"FADH
2
"
HydraEon"
DehydrogenaEon""
to"give"NADH

16. The$Citric$Acid$Cycle$$
""
• Per$each$turn$of$the$cycle:$
‒ One"acetyl"group"enters"(2"C)"and"2"CO
2
"leave"
‒ One"molecule"of"oxaloacetate"is"used"to"make"citrate"and"one"molecule"
is"regenerated"(no"net"change"in"OA"concentraEon;"which"is"very"low)"
‒ 4"of"the"8"steps"are"oxidaEons"(the"energy"of"oxidaEon"is"conserved"in"
NADH"and"FADH
2
)$
• Not$limited$to$energy$produc>on$$
‒ 4D"and"5DC"intermediates"serve"as"precursors"for"different"products"
‒ To"replace"these"intermediates,"cells"use"anaplero*c"(replenishing)"
reacEons"$

17. CAC$Bond$Forma>on$by$Condensa>on$of$
AcetylACoA$and$Oxaloacetate$(step$1)$
• CondensaEon"of"acetylDCoA"
"and"oxaloacetate"
• The"only"reacEon"with""
CDC"bond"formaEon"
• Uses"Acid/Base"Catalysis"
– Carbonyl of oxaloacetate
is a good electrophile (stabilization of carbanions)
– Methyl group has been converted to methylene
• Rate5limi*ng,step,of,CAC,
• AcEvity"largely"depends"on"[oxaloacetate]"
• Highly"thermodynamically"favorable/irreversible"
– Regulated"by"substrate"availability"and"product"inhibiEon"

18. Induced$Fit$in$the$Citrate$Synthase$
• oxaloacetate"binds"first"!"creaEng"a"binding"site"for"
acetylDCoA"
• Avoids"unnecessary"hydrolysis"of"thioester"in"acetylD
CoA"
"
"
b)$$Closed$conforma>on:$
Binding"of"OAA"creates"
binding"for"acetylDCoA"
ReacEve"carbanion"is"
protected""
a) Open$conforma>on:$
Free"enzyme"does"not"have"
a"binding"site"for"acetylDCoA"

19. Isomeriza>on$by$Dehydra>on/
Rehydra>on$(step$2)$

20. Aconitase$
• EliminaEon"of"H
2O"from"citrate"gives"a"cis"C=C"bond""
– Lyase"
• Citrate,"a"terEary"alcohol,"is"a"poor"substrate"for"oxidaEon"
• Isocitrate,"a"secondary"alcohol,"is"a"good"substrate"for"oxidaEon"
• AddiEon"of"H
2O"to"cisDaconitate"is"stereospecific"
(either"to"form"isocitrate"or"citrate)"
• Cytosolic"isozyme"uses"NADP
+
"as"a"cofactor"
• Thermodynamically"unfavorable/reversible"
– Product"is"consumed"rapidly"by"the"next"step"(concentraEon"
kept"low)"to"pull"reacEon"forward"

21. IronASulfur$Center$in$Aconitase$
• Water"removal"from"citrate"and"subsequent"addiEon"to"cisD
aconitate"are"catalyzed"by"the"ironDsulfur"center:"sensiEve"to"
oxidaEve"stress."
• The"ironDsulfur"center"acts"in"both"substrate"binding"and"catalysis."""

22. Aconitase$is$a$“moonligh>ng”$enzyme$
• When"Fe"is"deficient,"aconitase"loses"its"FeDS"center"and"acquires"
a"new%role"in"Fe"homeostasis"
• Cytosolic"Aconitase"is"an"enzyme"(with"FeDS)"and"a"regulator"of"
protein"synthesis"("–"Fe)"
• In"humans"Fe"levels"must"be"regulated:"too"liKle"!"anemia;"too"
much"!"liver"damage"
• Transferrin:"carries"Fe"in"the"blood"
• Transferrin,receptor:"receives"and"endocytoses"Fe"
• Ferri*n:"stores"excess"Fe"inside"the"cells"
• Apoaconitase"("–"Fe)"regulates"protein"levels"by"stabilizing"or"
destabilizing"the"mRNA"of"transferrin"receptor"or"ferriEn"""
• Apoaconitase"!""ferriEn"and"#"TfR"synthesis"(the"results"
would"be"an"increase"in"cellular"[Fe])"

23. Oxida>ve$Decarboxyla>on$#2$(step$3)$
• Carbon"is"lost"as"CO
2"
and"NADH"is"generated"
• Carbon"lost"as"CO
2
"did"NOT"come"from"acetylDCoA"
• OxidaEon"of"the"alcohol"to"a"ketone"
• Transfers"a"hydride"to"NAD
+
"
• Cytosolic"isozyme"uses"NADP
+
"as"a"cofactor"
• Highly"thermodynamically"favorable/irreversible"
• Regulated"by"product"inhibiEon"and"ATP"

24. Final$Oxida>ve$Decarboxyla>on$(step$4)$
• Last"oxidaEve""
decarboxylaEon"
– Net"full"oxidaEon"of"all""
carbons"of"glucose"
• SuccinylDCoA"is"another"higherDenergy"thioester"
bond"
• Highly"thermodynamically"favorable/irreversible"
– Regulated"by"product"inhibiEon"

25. Origin$of$CAatoms$in$CO
2
$
Both$CO
2
$carbon$atoms$are$derived$from$oxaloacetate$
We have lost 2 CO
2
already, so we have a net complete oxidation of
glucose after two pyruvates go through the CAC.

But its not the actual carbons from pyruvate (in red) in each cycle.
H
2C
C
H
2C
HO
COOH
COOH
COOH
Citrate Isocitrate
H
2C
HC
C
H
COOH
COOH
COOH
HO
α-ketoglutarate
H
2C
CH
2
C
COOH
COOHO
Succinyl-CoA
H
2C
CH
2
C
COOH
SCoAO

26. αAKetoglutarate$Dehydrogenase$
• Complex"similar"to"pyruvate"dehydrogenase"
– Same"coenzymes,"idenEcal"mechanisms"
– AcEve"sites"different"to"accommodate"differentDsized"substrates"
– E
1
"aa"sequences"differ"(and"specificity)"
E
2
"are"very"similar"
E
3
"are"idenEcal""

27. Genera>on$of$GTP$through$Thioester$(step$5)$
• Substrate"level""
phosphorylaEon"
• Energy"of"thioester"allows""
for"incorporaEon"of"inorganic"phosphate"into"
ADP"or"GDP"to"make"ATP"or"GTP"
• Goes"through"a"phosphoDenzyme"intermediate"
• Produces"GTP,"which"can"be"converted"to"ATP,"or"
ATP"directly"(2"isozymes"in"animal"cells,"specific"for"GDP"or"
ADP)"
• Slightly"thermodynamically"favorable/reversible"
– Product"concentraEon"kept"low"to"pull"forward"

28. Oxida>on$of$an$Alkane$to$Alkene$(step$6)$
• Bound"to"mitochondrial""
inner"membrane""
– Complex"II"in"the"ETC"
• OxidaEon"of"the"alkane"to"alkene"requires"FAD"
• FAD"is"covalently"bound"
• 3"FeDS"clusters"
• Near"equilibrium/reversible"
– Product"concentraEon"kept"low"to"pull"forward"

29. Hydra>on$Across$a$Double$Bond$(step$7)$
• Highly"stereospecific"
– AddiEon"of"water"is"always"trans"and""
forms"LDmalate"
– Cannot"work"on"maleate"(cis"isomer"of"fumarate)"
– OHD"adds"to"fumarate…"then"H+"adds"to"the"carbanion"
– Cannot"disEnguish"between"inner"carbons,"so"either"can"
gain"–OH"
• Slightly"thermodynamically"favorable/reversible"
– Product"concentraEon"kept"low"to"pull"reacEon"forward"

30. Oxida>on$of$Alcohol$to$a$Ketone$(step$8)$
• Final"step"of"the"cycle"
• Regenerates"oxaloacetate""
for"citrate"synthase"
• Highly"thermodynamically"UNfavorable/reversible"
– Oxaloacetate"concentraEon"kept"VERY"low"by"citrate"synthase"
("<"10
–6
"M)""
• Pulls"the"reacEon"forward"

31. One$Turn$of$the$Citric$Acid$Cycle$

32. Net$Result$of$the$Citric$Acid$Cycle$
• Net"oxidaEon"of"two"carbons"to"CO
2
"
– Equivalent"to"two"carbons"of"acetylDCoA""
– but"NOT"the"exact"same"carbons"
• Energy"captured"by"electron"transfer"to""NADH"and"
FADH
2
"
• Generates"1"GTP,"which"can"be"converted"to"ATP"
AcetylDCoA"+"3NAD
+
"+"FAD"+"GDP"+"P
i
"+"2"H
2O"!"
"""2CO
2
"+"3NADH"+"FADH
2"+"GTP"+"CoA"+"3H
+"

33. Direct$and$Indirect$ATP$Yield$

35. CAC$is$an$amphibolic$pathway$
• Amphibolic5,
serves"in"both"
catabolism"and"
anabolism"
• Precursors"for"
many"molecules"
• Needs"to"be"
replenished"(by"
anapleroEc"
reacEons)"
""""""Red%arrows%%

36. Anaplero>c$Reac>ons$
• Must"replenish"the"intermediates"in"order"for"the"
cycle"and"central"metabolic"pathway"to"conEnue"
• 4Dcarbon"intermediates"are"formed"by"carboxylaEon"
of"3Dcarbon"precursors"
• The"replenishing"and"consuming"reacEons"are"in"
dynamic"balance"([CAC"intermediates]"is"~"constant)"

37. Anaplero>c$Reac>ons$
• Must"replenish"the"intermediates"in"order"for"the"
cycle"and"central"metabolic"pathway"to"conEnue"
• 4Dcarbon"intermediates"are"formed"by"carboxylaEon"
of"3Dcarbon"precursors"
• The"replenishing"and"consuming"reacEons"are"in"
dynamic"balance"([CAC"intermediates]"is"~"constant)"
• Regulatory"enzyme"–"inac*ve"in"the"absence"of"
acetylDCoA"
• More"acetylDCoA,"more"acEvity"$"more"OAA"to"
react"with"acetylDCoA"to"start"the"cycle"

38. Bio>n$is$a$CO
2
$carrier$
• Vitamin"B7"(bioEn)"is"required"in"our"food"
• It"is"a"cofactor"(prostheEc"group)"in"carboxylases""
• BioEn"is"a"specialized"carrier"of"1DC"groups"in"their"
most"OXIDIZED"state"(CO
2
)"
• Pyruvate"carboxylase"has"4"idenEcal"subunits"each"
carrying"a"molecule"of"bioEn"
• It"is"present"in"many"foods"and"intesEnal""
bacteria"are"able"to"synthesize"it,"hence""
bioEn"deficiency"is"rare"
• ConsumpEon"of"raw"eggs"in"large"quanEEes"leads"
to"bioEn"deficiency"since"egg"white"have"the"
protein"avidin$which"binds"bioEn"very"Eghtly"and"
prevents"its"absorpEon"in"the"intesEne"

39. Biological$tethers$allow$flexibility$
• All"enter"the"cells"on"
the"same"transporter"
• All"are"covalently"
aKached"to"proteins"
• All"provide"flexible"
arms"on"the"enzymes"
to"which"they"are"
covalently"bound"
• All"act"as"tethers"that"
move"intermediates"
from"one"acEve"site"to"
the"next"

40. Regula>on$of$the$Citric$Acid$Cycle$
Cell"has"high"energy"
demands!"
Cell"is"supplied"with"
enough"energy"
[NADH]/[NAD
+
]"and"
[ATP]/[ADP]"are"
high"

41. Regula>on$of$the$Citric$Acid$Cycle$
• Regulated"at"highly"thermodynamically"favorable"
and"irreversible"steps"
– PDH,"citrate"synthase,"IDH,"and"αKDH"
• General"regulatory"mechanism"
– AcEvated"by"substrate"availability"
– Inhibited"by"product"accumulaEon"
– Overall"products"of"the"pathway"are"NADH"and"ATP"
• Affect"all"regulated"enzymes"in"the"cycle"
• Inhibitors:"NADH"and"ATP"
• AcEvators:"NAD
+
"and"AMP"
• Ca
2+
"in"muscles"acEvates"the"cycle"(Ca
2+
"signals"muscle"
contracEon"$"need"for"energy)"

42. Regula>on$of$Pyruvate$Dehydrogenase$
• Also"regulated"by"reversible"phosphorylaEon"of"E1"
– PhosphorylaEon:"inacEve"
– DephosphorylaEon:"acEve"
• PDH"kinase"and"PDH"phosphatase"are"part"of"
mammalian"PDH"complex"
– Kinase"is"acEvated"by"ATP""
• High"ATP"!"phosphorylated"PDH"!"less"acetylDCoA"
• Low"ATP"!"kinase"is"less"acEve"and"phosphatase"
removes"phosphate"from"PDH"!"more"acetylDCoA"
– Phosphatase"is"acEvated"by"insulin,"PEP,"and"AMP,"
and"inhibited"by"ATP,"NADH,"and"AcetylDCoA"

43. Addi>onal$Regulatory$Mechanisms$
• Citrate"synthase"is"also"inhibited"by"succinylDCoA"
– αDketoglutarate"is"an"important"branch"point"for"amino"
acid"metabolism"
– SuccinylDCoA"communicates"flow"at"this"branch"point"to"
the"start"of"the"cycle"
• RegulaEon"of"isocitrate"dehydrogenase"controls"
citrate"levels"
– Aconitase"is"reversible"
– InhibiEon"of"IDH"leads"to"accumulaEon"of"isocitrate"and"
reverses"aconitase"
– Accumulated"citrate"leaves"mitochondria"and"inhibits"
PFKD1"in"glycolysis"

44. CAC$muta>ons$lead$to$cancer$
• MutaEons"in"CAC"enzymes"are"very"rare"in"
man"
• GeneEc"defects"in"fumarase"!"smooth"
muscle"and"kidney"cancer"
• MutaEons"in"succinate"DH"!"tumors"of"the"
adrenal"glands"
• Both"enzymes"are"defined"as"tumor"
suppressor"genes"
• IDH"mutaEon"leads"to"a"new"funcEon"of"the"
enzyme"the"net"result"of"which"is"the"
development"of"glial"cell"tumors"in"the"brain""

45. Ques>on$5$(Take$home$exam)$$
Due:$NEXT$WEEK$(js>[email protected])$
• Please$solve$ques>ons:$
1. 5$(NAD$redox$carriers)$
2. 10$(OAA$in$mito)$
3. 18$(
14
CAglucose)$
4. 19$(Beriberi)$
For%wri9en%answers,%I%prefer%to%have%them%typed%in%Word.%I%
can%accept%the%assignment%in%one%file%sent%to%my%email.%For%
answers%that%require%solving%mathema3cally,%you%can%either%
type%them%or%write%them%down%and%scan%them.%