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16|	
  The	
  Citric	
  Acid	
  Cycle	
  
© 2013 W. H. Freeman and Company
Only	
  a	
  small	
  amount	
  of	
  energy	
  available	
  
in	
  glucose	
  is	
  captured	
  in	
  glycolysis	
  
2
ΔG′° = –146 kJ/mol
Glycolysis
Full oxidation (+ 6 O2)
ΔG′° = –2,840 kJ/mol
6 CO2 + 6 H2O
Glucose
Only the 1st stage of
Glucose oxidation
Cellular	
  Respira>on	
  
• 	
  Process	
  in	
  which	
  cells	
  consume	
  O2	
  and	
  produce	
  CO2	
  
• 	
  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:	
  	
  
-­‐ 	
  acetyl	
  CoA	
  producEon	
  (from	
  organic	
  fuel	
  molecules)	
  
-­‐ 	
  acetyl	
  CoA	
  oxidaEon	
  (in	
  the	
  CAC	
  to	
  produce	
  CO2)	
  
-­‐ 	
  electron	
  transfer	
  and	
  oxidaEve	
  phosphorylaEon	
  
(reduced	
  coenzymes	
  from	
  CAC	
  give	
  their	
  e–’s	
  to	
  O2	
  
forming	
  ATP	
  in	
  the	
  process)	
  
Respira>on:	
  Stage	
  1	
  
Acetyl-­‐CoA	
  Produc>on	
  
•  AcEvated	
  form	
  of	
  acetate	
  
•  C-­‐skeleton	
  of	
  sugars	
  and	
  faKy	
  
acids	
  are	
  converted	
  to	
  acetyl-­‐
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)	
  	
  
Respira>on:	
  Stage	
  2	
  
Acetyl-­‐CoA	
  oxida>on	
  
Generates	
  
NADH,	
  FADH2,	
  
and	
  one	
  GTP	
  
Respira>on:	
  Stage	
  3	
  
Oxida>ve	
  Phosphoryla>on	
  
Generates	
  
a	
  lot	
  of	
  
ATP	
  
	
  
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	
  
Conversion	
  of	
  Pyruvate	
  to	
  Acetyl-­‐CoA	
  
•  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	
  CoA-­‐SH	
  	
  are	
  co-­‐substrates	
  
TPP	
  –	
  thiamine	
  (B1)	
  
FAD	
  –	
  	
  riboflavin	
  (B2)	
  
NAD	
  –	
  niacin	
  (B3)	
  
CoA	
  –	
  panthothenic	
  acid	
  (B5)	
  
Derived	
  from:	
  
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)	
  
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	
  
Pyruvate	
  Dehydrogenase	
  Complex	
  (PDH)	
  
• 	
  Large	
  (up	
  to	
  10	
  MDa)	
  mulEenzyme	
  complex	
  	
  
-­‐ 	
  large	
  enough	
  to	
  be	
  seen	
  with	
  cryoEM	
  
-­‐ 	
  pyruvate	
  dehydrogenase	
  (E1)	
  
-­‐ 	
  dihydrolipoyl	
  transacetylase	
  (E2)	
  
-­‐ 	
  dihydrolipoyl	
  dehydrogenase	
  (E3)	
  
-­‐ 	
  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	
  
3D	
  Reconstruc>on	
  from	
  Cryo-­‐EM	
  data	
  
The	
  bovine	
  enzyme	
  
Core:	
  60	
  idenEcal	
  	
  
copies	
  of	
  E2)	
  
E1	
  acEve	
  site	
  has	
  	
  
bound	
  TPP	
  
E3	
  acEve	
  site	
  has	
  	
  
bound	
  FAD	
  
Protein	
  kinase	
  and	
  phosphoprotein	
  phosphatase	
  are	
  part	
  of	
  the	
  complex	
  
Overall	
  Reac>on	
  of	
  PDH	
  
E1	
  	
  
• Step	
  1:	
  DecarboxylaEon	
  of	
  pyruvate	
  to	
  an	
  aldehyde	
  	
  
• Step	
  2:	
  OxidaEon	
  of	
  aldehyde	
  to	
  a	
  carboxylic	
  acid	
  
‒  Electrons	
  reduce	
  lipoamide	
  and	
  form	
  a	
  thioester	
  
E2	
  
• 	
  Step	
  3:	
  	
  
FormaEon	
  	
  
of	
  acetyl-­‐CoA	
  	
  
(product	
  1)	
  
E3	
  	
  
• Step	
  4:	
  	
  
ReoxidaEon	
  of	
  the	
  lipoamide	
  cofactor	
  
• Step	
  5:	
  	
  
RegeneraEon	
  of	
  the	
  oxidized	
  FAD	
  cofactor	
  
‒  Forming	
  NADH	
  (product	
  2)	
  
Chapter 16 - The citric acid cycle - Biochemistry
The	
  Citric	
  Acid	
  Cycle	
  (CAC)	
  
C-­‐C	
  bond	
  formaEon	
  to	
  	
  
make	
  citrate	
  	
  
IsomerizaEon	
  via	
  	
  
dehyd./rehydraEon
OxidaEve	
  decarboxy-­‐	
  
laEons	
  to	
  give	
  2	
  NADH
Substrate-­‐level	
  phosphorylaEon	
  to	
  give	
  GTP
DehydrogenaEon	
  	
  
to	
  give	
  	
  
reduced	
  FADH2	
  
HydraEon	
  
DehydrogenaEon	
  	
  
to	
  give	
  NADH
The	
  Citric	
  Acid	
  Cycle	
  	
  
	
  	
  
• Per	
  each	
  turn	
  of	
  the	
  cycle:	
  
‒  One	
  acetyl	
  group	
  enters	
  (2	
  C)	
  and	
  2	
  CO2	
  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	
  FADH2)	
  
• Not	
  limited	
  to	
  energy	
  produc>on	
  	
  
‒  4-­‐	
  and	
  5-­‐C	
  intermediates	
  serve	
  as	
  precursors	
  for	
  different	
  products	
  
‒  To	
  replace	
  these	
  intermediates,	
  cells	
  use	
  anaplero*c	
  (replenishing)	
  
reacEons	
  	
  
C-­‐C	
  Bond	
  Forma>on	
  by	
  Condensa>on	
  of	
  
Acetyl-­‐CoA	
  and	
  Oxaloacetate	
  (step	
  1)	
  
•  CondensaEon	
  of	
  acetyl-­‐CoA	
  
	
  and	
  oxaloacetate	
  
•  The	
  only	
  reacEon	
  with	
  	
  
C-­‐C	
  bond	
  formaEon	
  
•  Uses	
  Acid/Base	
  Catalysis	
  
–  Carbonyl of oxaloacetate
is a good electrophile (stabilization of carbanions)
–  Methyl group has been converted to methylene
•  Rate-­‐limi*ng	
  step	
  of	
  CAC	
  
•  AcEvity	
  largely	
  depends	
  on	
  [oxaloacetate]	
  
•  Highly	
  thermodynamically	
  favorable/irreversible	
  
–  Regulated	
  by	
  substrate	
  availability	
  and	
  product	
  inhibiEon	
  
Induced	
  Fit	
  in	
  the	
  Citrate	
  Synthase	
  
•  oxaloacetate	
  binds	
  first	
  à	
  creaEng	
  a	
  binding	
  site	
  for	
  
acetyl-­‐CoA	
  
•  Avoids	
  unnecessary	
  hydrolysis	
  of	
  thioester	
  in	
  acetyl-­‐
CoA	
  
	
  
	
  
b)	
  	
  Closed	
  conforma>on:	
  
Binding	
  of	
  OAA	
  creates	
  
binding	
  for	
  acetyl-­‐CoA	
  
ReacEve	
  carbanion	
  is	
  
protected	
  	
  
a)  Open	
  conforma>on:	
  
Free	
  enzyme	
  does	
  not	
  have	
  
a	
  binding	
  site	
  for	
  acetyl-­‐CoA	
  
Isomeriza>on	
  by	
  Dehydra>on/
Rehydra>on	
  (step	
  2)	
  
Aconitase	
  
•  EliminaEon	
  of	
  H2O	
  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	
  H2O	
  to	
  cis-­‐aconitate	
  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	
  
Iron-­‐Sulfur	
  Center	
  in	
  Aconitase	
  
•  Water	
  removal	
  from	
  citrate	
  and	
  subsequent	
  addiEon	
  to	
  cis-­‐
aconitate	
  are	
  catalyzed	
  by	
  the	
  iron-­‐sulfur	
  center:	
  sensiEve	
  to	
  
oxidaEve	
  stress.	
  
•  The	
  iron-­‐sulfur	
  center	
  acts	
  in	
  both	
  substrate	
  binding	
  and	
  catalysis.	
  	
  	
  
Aconitase	
  is	
  a	
  “moonligh>ng”	
  enzyme	
  
•  When	
  Fe	
  is	
  deficient,	
  aconitase	
  loses	
  its	
  Fe-­‐S	
  center	
  and	
  acquires	
  
a	
  new	
  role	
  in	
  Fe	
  homeostasis	
  
•  Cytosolic	
  Aconitase	
  is	
  an	
  enzyme	
  (with	
  Fe-­‐S)	
  and	
  a	
  regulator	
  of	
  
protein	
  synthesis	
  (	
  –	
  Fe)	
  
•  In	
  humans	
  Fe	
  levels	
  must	
  be	
  regulated:	
  too	
  liKle	
  à	
  anemia;	
  too	
  
much	
  à	
  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	
  à	
  êferriEn	
  and	
  é	
  TfR	
  synthesis	
  (the	
  results	
  
would	
  be	
  an	
  increase	
  in	
  cellular	
  [Fe])	
  
Oxida>ve	
  Decarboxyla>on	
  #2	
  (step	
  3)	
  
•  Carbon	
  is	
  lost	
  as	
  CO2	
  and	
  NADH	
  is	
  generated	
  
•  Carbon	
  lost	
  as	
  CO2	
  did	
  NOT	
  come	
  from	
  acetyl-­‐CoA	
  
•  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	
  
Final	
  Oxida>ve	
  Decarboxyla>on	
  (step	
  4)	
  
•  Last	
  oxidaEve	
  	
  
decarboxylaEon	
  
– Net	
  full	
  oxidaEon	
  of	
  all	
  	
  
carbons	
  of	
  glucose	
  
•  Succinyl-­‐CoA	
  is	
  another	
  higher-­‐energy	
  thioester	
  
bond	
  
•  Highly	
  thermodynamically	
  favorable/irreversible	
  
– Regulated	
  by	
  product	
  inhibiEon	
  
Origin	
  of	
  C-­‐atoms	
  in	
  CO2	
  
Both	
  CO2	
  carbon	
  atoms	
  are	
  derived	
  from	
  oxaloacetate	
  
We have lost 2 CO2 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.
H2C
C
H2C
HO
COOH
COOH
COOH
Citrate Isocitrate
H2C
HC
C
H
COOH
COOH
COOH
HO
α-ketoglutarate
H2C
CH2
C
COOH
COOHO
Succinyl-CoA
H2C
CH2
C
COOH
SCoAO
α-­‐Ketoglutarate	
  Dehydrogenase	
  
•  Complex	
  similar	
  to	
  pyruvate	
  dehydrogenase	
  
–  Same	
  coenzymes,	
  idenEcal	
  mechanisms	
  
–  AcEve	
  sites	
  different	
  to	
  accommodate	
  different-­‐sized	
  substrates	
  
–  E1	
  aa	
  sequences	
  differ	
  (and	
  specificity)	
  
E2	
  are	
  very	
  similar	
  
E3	
  are	
  idenEcal	
  	
  
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	
  phospho-­‐enzyme	
  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	
  
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	
  Fe-­‐S	
  clusters	
  
•  Near	
  equilibrium/reversible	
  
–  Product	
  concentraEon	
  kept	
  low	
  to	
  pull	
  forward	
  
Hydra>on	
  Across	
  a	
  Double	
  Bond	
  (step	
  7)	
  
•  Highly	
  stereospecific	
  
–  AddiEon	
  of	
  water	
  is	
  always	
  trans	
  and	
  	
  
forms	
  L-­‐malate	
  
–  Cannot	
  work	
  on	
  maleate	
  (cis	
  isomer	
  of	
  fumarate)	
  
–  OH-­‐	
  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	
  
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	
  
One	
  Turn	
  of	
  the	
  Citric	
  Acid	
  Cycle	
  
Net	
  Result	
  of	
  the	
  Citric	
  Acid	
  Cycle	
  
•  Net	
  oxidaEon	
  of	
  two	
  carbons	
  to	
  CO2	
  
– Equivalent	
  to	
  two	
  carbons	
  of	
  acetyl-­‐CoA	
  	
  
– but	
  NOT	
  the	
  exact	
  same	
  carbons	
  
•  Energy	
  captured	
  by	
  electron	
  transfer	
  to	
  	
  NADH	
  and	
  
FADH2	
  
•  Generates	
  1	
  GTP,	
  which	
  can	
  be	
  converted	
  to	
  ATP	
  
Acetyl-­‐CoA	
  +	
  3NAD+	
  +	
  FAD	
  +	
  GDP	
  +	
  Pi	
  +	
  2	
  H2O	
  à	
  
	
   	
  	
  2CO2	
  +	
  3NADH	
  +	
  FADH2	
  +	
  GTP	
  +	
  CoA	
  +	
  3H+	
  
Direct	
  and	
  Indirect	
  ATP	
  Yield	
  
Chapter 16 - The citric acid cycle - Biochemistry
CAC	
  is	
  an	
  amphibolic	
  pathway	
  
•  Amphibolic-­‐	
  
serves	
  in	
  both	
  
catabolism	
  and	
  
anabolism	
  
•  Precursors	
  for	
  
many	
  molecules	
  
•  Needs	
  to	
  be	
  
replenished	
  (by	
  
anapleroEc	
  
reacEons)	
  
	
  	
  	
  	
  	
  	
  Red	
  arrows	
  	
  
Anaplero>c	
  Reac>ons	
  
•  Must	
  replenish	
  the	
  intermediates	
  in	
  order	
  for	
  the	
  
cycle	
  and	
  central	
  metabolic	
  pathway	
  to	
  conEnue	
  
•  4-­‐carbon	
  intermediates	
  are	
  formed	
  by	
  carboxylaEon	
  
of	
  3-­‐carbon	
  precursors	
  
•  The	
  replenishing	
  and	
  consuming	
  reacEons	
  are	
  in	
  
dynamic	
  balance	
  ([CAC	
  intermediates]	
  is	
  ~	
  constant)	
  
Anaplero>c	
  Reac>ons	
  
•  Must	
  replenish	
  the	
  intermediates	
  in	
  order	
  for	
  the	
  
cycle	
  and	
  central	
  metabolic	
  pathway	
  to	
  conEnue	
  
•  4-­‐carbon	
  intermediates	
  are	
  formed	
  by	
  carboxylaEon	
  
of	
  3-­‐carbon	
  precursors	
  
•  The	
  replenishing	
  and	
  consuming	
  reacEons	
  are	
  in	
  
dynamic	
  balance	
  ([CAC	
  intermediates]	
  is	
  ~	
  constant)	
  
•  Regulatory	
  enzyme	
  –	
  inac*ve	
  in	
  the	
  absence	
  of	
  
acetyl-­‐CoA	
  
•  More	
  acetyl-­‐CoA,	
  more	
  acEvity	
  è	
  more	
  OAA	
  to	
  
react	
  with	
  acetyl-­‐CoA	
  to	
  start	
  the	
  cycle	
  
Bio>n	
  is	
  a	
  CO2	
  carrier	
  
•  Vitamin	
  B7	
  (bioEn)	
  is	
  required	
  in	
  our	
  food	
  
•  It	
  is	
  a	
  cofactor	
  (prostheEc	
  group)	
  in	
  carboxylases	
  	
  
•  BioEn	
  is	
  a	
  specialized	
  carrier	
  of	
  1-­‐C	
  groups	
  in	
  their	
  
most	
  OXIDIZED	
  state	
  (CO2)	
  
•  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	
  
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	
  
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	
  
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	
  
• Ca2+	
  in	
  muscles	
  acEvates	
  the	
  cycle	
  (Ca2+	
  signals	
  muscle	
  
contracEon	
  è	
  need	
  for	
  energy)	
  
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	
  à	
  phosphorylated	
  PDH	
  à	
  less	
  acetyl-­‐CoA	
  
• Low	
  ATP	
  à	
  kinase	
  is	
  less	
  acEve	
  and	
  phosphatase	
  
removes	
  phosphate	
  from	
  PDH	
  à	
  more	
  acetyl-­‐CoA	
  
–  Phosphatase	
  is	
  acEvated	
  by	
  insulin,	
  PEP,	
  and	
  AMP,	
  
and	
  inhibited	
  by	
  ATP,	
  NADH,	
  and	
  Acetyl-­‐CoA	
  
Addi>onal	
  Regulatory	
  Mechanisms	
  
•  Citrate	
  synthase	
  is	
  also	
  inhibited	
  by	
  succinyl-­‐CoA	
  
–  α-­‐ketoglutarate	
  is	
  an	
  important	
  branch	
  point	
  for	
  amino	
  
acid	
  metabolism	
  
–  Succinyl-­‐CoA	
  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	
  
PFK-­‐1	
  in	
  glycolysis	
  
CAC	
  muta>ons	
  lead	
  to	
  cancer	
  
•  MutaEons	
  in	
  CAC	
  enzymes	
  are	
  very	
  rare	
  in	
  
man	
  
•  GeneEc	
  defects	
  in	
  fumarase	
  à	
  smooth	
  
muscle	
  and	
  kidney	
  cancer	
  
•  MutaEons	
  in	
  succinate	
  DH	
  à	
  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	
  	
  
Ques>on	
  5	
  (Take	
  home	
  exam)	
  	
  
Due:	
  NEXT	
  WEEK	
  (js>ban@birzeit.edu)	
  
•  Please	
  solve	
  ques>ons:	
  
1.  5	
  (NAD	
  redox	
  carriers)	
  
2.  10	
  (OAA	
  in	
  mito)	
  
3.  18	
  (14C-­‐glucose)	
  
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.	
  

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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 O2) ΔG′° = –2,840 kJ/mol 6 CO2 + 6 H2O Glucose Only the 1st stage of Glucose oxidation
  • 3. Cellular  Respira>on   •   Process  in  which  cells  consume  O2  and  produce  CO2   •   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:     -­‐   acetyl  CoA  producEon  (from  organic  fuel  molecules)   -­‐   acetyl  CoA  oxidaEon  (in  the  CAC  to  produce  CO2)   -­‐   electron  transfer  and  oxidaEve  phosphorylaEon   (reduced  coenzymes  from  CAC  give  their  e–’s  to  O2   forming  ATP  in  the  process)  
  • 4. Respira>on:  Stage  1   Acetyl-­‐CoA  Produc>on   •  AcEvated  form  of  acetate   •  C-­‐skeleton  of  sugars  and  faKy   acids  are  converted  to  acetyl-­‐ 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   Acetyl-­‐CoA  oxida>on   Generates   NADH,  FADH2,   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  Acetyl-­‐CoA   •  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  CoA-­‐SH    are  co-­‐substrates   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     -­‐   large  enough  to  be  seen  with  cryoEM   -­‐   pyruvate  dehydrogenase  (E1)   -­‐   dihydrolipoyl  transacetylase  (E2)   -­‐   dihydrolipoyl  dehydrogenase  (E3)   -­‐   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  Cryo-­‐EM  data   The  bovine  enzyme   Core:  60  idenEcal     copies  of  E2)   E1  acEve  site  has     bound  TPP   E3  acEve  site  has     bound  FAD   Protein  kinase  and  phosphoprotein  phosphatase  are  part  of  the  complex  
  • 13. Overall  Reac>on  of  PDH   E1     • Step  1:  DecarboxylaEon  of  pyruvate  to  an  aldehyde     • Step  2:  OxidaEon  of  aldehyde  to  a  carboxylic  acid   ‒  Electrons  reduce  lipoamide  and  form  a  thioester   E2   •   Step  3:     FormaEon     of  acetyl-­‐CoA     (product  1)   E3     • 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)   C-­‐C  bond  formaEon  to     make  citrate     IsomerizaEon  via     dehyd./rehydraEon OxidaEve  decarboxy-­‐   laEons  to  give  2  NADH Substrate-­‐level  phosphorylaEon  to  give  GTP DehydrogenaEon     to  give     reduced  FADH2   HydraEon   DehydrogenaEon     to  give  NADH
  • 16. The  Citric  Acid  Cycle         • Per  each  turn  of  the  cycle:   ‒  One  acetyl  group  enters  (2  C)  and  2  CO2  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  FADH2)   • Not  limited  to  energy  produc>on     ‒  4-­‐  and  5-­‐C  intermediates  serve  as  precursors  for  different  products   ‒  To  replace  these  intermediates,  cells  use  anaplero*c  (replenishing)   reacEons    
  • 17. C-­‐C  Bond  Forma>on  by  Condensa>on  of   Acetyl-­‐CoA  and  Oxaloacetate  (step  1)   •  CondensaEon  of  acetyl-­‐CoA    and  oxaloacetate   •  The  only  reacEon  with     C-­‐C  bond  formaEon   •  Uses  Acid/Base  Catalysis   –  Carbonyl of oxaloacetate is a good electrophile (stabilization of carbanions) –  Methyl group has been converted to methylene •  Rate-­‐limi*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  à  creaEng  a  binding  site  for   acetyl-­‐CoA   •  Avoids  unnecessary  hydrolysis  of  thioester  in  acetyl-­‐ CoA       b)    Closed  conforma>on:   Binding  of  OAA  creates   binding  for  acetyl-­‐CoA   ReacEve  carbanion  is   protected     a)  Open  conforma>on:   Free  enzyme  does  not  have   a  binding  site  for  acetyl-­‐CoA  
  • 20. Aconitase   •  EliminaEon  of  H2O  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  H2O  to  cis-­‐aconitate  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. Iron-­‐Sulfur  Center  in  Aconitase   •  Water  removal  from  citrate  and  subsequent  addiEon  to  cis-­‐ aconitate  are  catalyzed  by  the  iron-­‐sulfur  center:  sensiEve  to   oxidaEve  stress.   •  The  iron-­‐sulfur  center  acts  in  both  substrate  binding  and  catalysis.      
  • 22. Aconitase  is  a  “moonligh>ng”  enzyme   •  When  Fe  is  deficient,  aconitase  loses  its  Fe-­‐S  center  and  acquires   a  new  role  in  Fe  homeostasis   •  Cytosolic  Aconitase  is  an  enzyme  (with  Fe-­‐S)  and  a  regulator  of   protein  synthesis  (  –  Fe)   •  In  humans  Fe  levels  must  be  regulated:  too  liKle  à  anemia;  too   much  à  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  à  êferriEn  and  é  TfR  synthesis  (the  results   would  be  an  increase  in  cellular  [Fe])  
  • 23. Oxida>ve  Decarboxyla>on  #2  (step  3)   •  Carbon  is  lost  as  CO2  and  NADH  is  generated   •  Carbon  lost  as  CO2  did  NOT  come  from  acetyl-­‐CoA   •  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   •  Succinyl-­‐CoA  is  another  higher-­‐energy  thioester   bond   •  Highly  thermodynamically  favorable/irreversible   – Regulated  by  product  inhibiEon  
  • 25. Origin  of  C-­‐atoms  in  CO2   Both  CO2  carbon  atoms  are  derived  from  oxaloacetate   We have lost 2 CO2 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. H2C C H2C HO COOH COOH COOH Citrate Isocitrate H2C HC C H COOH COOH COOH HO α-ketoglutarate H2C CH2 C COOH COOHO Succinyl-CoA H2C CH2 C COOH SCoAO
  • 26. α-­‐Ketoglutarate  Dehydrogenase   •  Complex  similar  to  pyruvate  dehydrogenase   –  Same  coenzymes,  idenEcal  mechanisms   –  AcEve  sites  different  to  accommodate  different-­‐sized  substrates   –  E1  aa  sequences  differ  (and  specificity)   E2  are  very  similar   E3  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  phospho-­‐enzyme  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  Fe-­‐S  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  L-­‐malate   –  Cannot  work  on  maleate  (cis  isomer  of  fumarate)   –  OH-­‐  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  CO2   – Equivalent  to  two  carbons  of  acetyl-­‐CoA     – but  NOT  the  exact  same  carbons   •  Energy  captured  by  electron  transfer  to    NADH  and   FADH2   •  Generates  1  GTP,  which  can  be  converted  to  ATP   Acetyl-­‐CoA  +  3NAD+  +  FAD  +  GDP  +  Pi  +  2  H2O  à        2CO2  +  3NADH  +  FADH2  +  GTP  +  CoA  +  3H+  
  • 33. Direct  and  Indirect  ATP  Yield  
  • 35. CAC  is  an  amphibolic  pathway   •  Amphibolic-­‐   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   •  4-­‐carbon  intermediates  are  formed  by  carboxylaEon   of  3-­‐carbon  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   •  4-­‐carbon  intermediates  are  formed  by  carboxylaEon   of  3-­‐carbon  precursors   •  The  replenishing  and  consuming  reacEons  are  in   dynamic  balance  ([CAC  intermediates]  is  ~  constant)   •  Regulatory  enzyme  –  inac*ve  in  the  absence  of   acetyl-­‐CoA   •  More  acetyl-­‐CoA,  more  acEvity  è  more  OAA  to   react  with  acetyl-­‐CoA  to  start  the  cycle  
  • 38. Bio>n  is  a  CO2  carrier   •  Vitamin  B7  (bioEn)  is  required  in  our  food   •  It  is  a  cofactor  (prostheEc  group)  in  carboxylases     •  BioEn  is  a  specialized  carrier  of  1-­‐C  groups  in  their   most  OXIDIZED  state  (CO2)   •  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   • Ca2+  in  muscles  acEvates  the  cycle  (Ca2+  signals  muscle   contracEon  è  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  à  phosphorylated  PDH  à  less  acetyl-­‐CoA   • Low  ATP  à  kinase  is  less  acEve  and  phosphatase   removes  phosphate  from  PDH  à  more  acetyl-­‐CoA   –  Phosphatase  is  acEvated  by  insulin,  PEP,  and  AMP,   and  inhibited  by  ATP,  NADH,  and  Acetyl-­‐CoA  
  • 43. Addi>onal  Regulatory  Mechanisms   •  Citrate  synthase  is  also  inhibited  by  succinyl-­‐CoA   –  α-­‐ketoglutarate  is  an  important  branch  point  for  amino   acid  metabolism   –  Succinyl-­‐CoA  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   PFK-­‐1  in  glycolysis  
  • 44. CAC  muta>ons  lead  to  cancer   •  MutaEons  in  CAC  enzymes  are  very  rare  in   man   •  GeneEc  defects  in  fumarase  à  smooth   muscle  and  kidney  cancer   •  MutaEons  in  succinate  DH  à  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>ban@birzeit.edu)   •  Please  solve  ques>ons:   1.  5  (NAD  redox  carriers)   2.  10  (OAA  in  mito)   3.  18  (14C-­‐glucose)   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.