atp is the cell’s “energy” but –cells also have….reducing power! processes (such as...
TRANSCRIPT
• ATP is the cell’s “energy” BUT– Cells also have….REDUCING POWER!
• Processes (such as photosynthesis) require NADPH as well as ATP
• NADH and NADPH are NOT interchangable
Pentose Phosphate Pathway
Hexose monophosphate (HMP) shunt / Phosphogluconate pathway.
NADH and NADPH are NOT interchangable
• NAD+ participates in synthesis of ATP • glycoloysis, oxidative phosphorylation
• NADPH is a reducing agent • produced in light reactions and consumed in Calvin
cycle of photosynthesis
NADP+ + 2H ---> NADPH + H+
In the cell…
[NAD+] ~ 1000 [NADP+]
~ 0.01[NADH] [NADPH]
1C5 to 1C5
2C5 to 2C5
2C5 to1C7 + 1C3
3C6 3C53C1
1C7 + 1C3 to1C6 + 1C4
1C4 + 1C5 to1C6 + 1C3
C
O
OH
OH
OH
CH2OPO32-
OC
O
OH
OH
OH
CH2OPO32-
O
H
H
B
N+
O
NH2
R
N
O
NH2
R
C
O
OH
OH
OH
CH2OPO32-
O
H
O HB
H
A
C
OH
OH
OH
OH
CH2OPO32-
O
OH
-O O
OHH
O
OH
OH
CH2OPO32-
H
H
OC
O
OHH
O-
OH
OH
CH2OPO32-
H
H
H
CH2OH
O
OH
OH
CH2OPO32-
H
H
A
Summary of carbon skeleton rearrangements in the pentose phosphate
pathway.
3C6 ---> 3C5 + 3C1
2
3
2
3 ribulose-5-P ---> 2 xylulose-5-P + 1 ribose-5-P
CH2OH
O
H
OH
CH2OPO32-
OH
H
H O
H OH
OHH
CH2OPO32-
OHH
CH2OH
O
HO
OH
CH2OPO32-
H
H
O
H
OH
CH2OPO32-
OH
H
H O
H OH
OHH
CH2OPO32-
OHH
H
H
OH
B
OH
H
OH
CH2OPO32-
OH
H
H O
H A
HB
HA
O
H
OH
CH2OPO32-
OH
H
H
H
OH
B O-
OH
CH2OPO32-
H
H
H
OH
HO H A
O
HO
OH
CH2OPO32-
H
H
H
H
OH
Transketolase: catalyzes the transfer of C2 units
CH2OH
O
HO
OH
CH2OPO32-
H
H
S
C-
N+H3C
R
R
CH2OH
OH
O
OH
CH2OPO32-
H
H
H A
S
C
N+H3C
R
R
H
B
CH2OH
OH
O
OH
CH2OPO32-
H
H
S
C
NH3C
R
R
CH2OH
OH
S
C
NH3C
R
R
H O
H OH
OHH
H
A
CH2OH
O
S
C
N+H3C
R
R
H OH
OHH
HO H
H
B
CH2OH
O
H OH
OHH
HO H
S
C-
N+X
R
R
CH2OPO32-
OHH
CH2OPO32-
CH2OPO32-
OHH
OHHC3
C7
CH3
Transaldolase: catalyzes the transfer of C3 units
CH2OH
O
H OH
OHH
HO H
CH2OPO32-
OHH
+H3NEnzyme
Schiff Base Formation
CH2OH
H O
OHH
HO H
CH2OPO32-
OHH
NH+
Enzyme
H B
CH2OH
H O
OHH
HO H
CH2OPO32-
OHH
NEnzyme
OH
CH2OPO32-
OHH
H A
CH2OH
HO H
NH+
Enzyme
OH
CH2OPO32-
OHH
H
Schiff BaseHydrolysis
CH2OH
HO H
O
OH
CH2OPO32-
OHH
H
C7
C4
C6C3
CH2OH
O
HO
OH
CH2OPO32-
H
H
S
C-
N+H3C
R
R
CH2OH
OH
O
OH
CH2OPO32-
H
H
H A
S
C
N+H3C
R
R
H
B
CH2OH
OH
O
OH
CH2OPO32-
H
H
S
C
NH3C
R
R
CH2OH
OH
S
C
NH3C
R
R
H O
H OH
OHH
CH2OPO32-
H
A
CH2OH
O
S
C
N+H3C
R
R
H OH
OHH
CH2OPO32-
HO H
H
B
CH2OH
O
H OH
OHH
CH2OPO32-
HO H
S
C-
N+H3C
R
R
C5
C3
C4
C6
Summary of the pentose phosphate pathway
3G6P + 6NADP+ + 3H2O
6NADPH + 6H+ + 3CO2 + 2F6P + GAP
Important intermediates
Ribose-5-phosphate (nucleic acids, histidine)Erythrose-4-phosphate (aromatic amino acids)
What is the purpose of the pentose phosphate pathway?
1) Biosynthetic precursors2) NADPH for biosynthesis3) NADPH to keep cell reduced
O2 + 2e- + 2H+ ----> H2O2
H2O2 + 2e- + 2H+ ----> 2H2O
O2 + 4H+ + 4e- ----> 2H2O
Eº (V) vs. NHE+0.28+1.35
+0.82
Oxygen Biochemistry
Reduction of O2 or H2O2 can be used as a thermodynamic driving force to drive oxidation
of various molecules
O2 + 2e- + 2H+ ----> H2O2
S ----> S2+ + 2e-
O2 + 4e- + 2H+ ----> 2H2O2S ----> 2S2+ + 4e-
H2O2 + 2e- + 2H+ ----> 2H2OS ----> S2+ + 2e-
Oxidative Damage
OHHO OH
OH
OH OH
HN
NH
O
S
CH3
O
SO32- S S
NH
NNH
HN
O
NH2
O
meta-tyrosine ortho tyrosine DOPA
dityrosine
2-oxohistidine
methionine sulfoxide cysteine sulfinatedisulfides
8-oxoguanosine
O
OH
lipid hydroperoxides
Peptide CleavagePhosphodiester cleavage
Peptide and phosphodiester cleavage
Iron-sulfur cluster disassembly
Oxygen Diradical
O O
1s
2s
2px 2py 2pz
1s
2s
2px 2py 2pz
2s
2s*
1s
1s*
2px
2px 2px
2px 2px
2px*
3O2(up/up) + 1X (paired) ---> 1XO2(paired)
1O2(paired) + 1X (paired) ---> 1XO2(paired)
3O2(up/up) + 3X (up/up) ---> 1XO2(paired)
Need to alleviate spin restriction
••O2 + e- ----> •O2-
•O2- + e- + 2H+ ----> H2O2
H2O2 + e- + H+ ----> H2O + •OH•OH + e- + H+ ----> H2O
••O2 + 2e- + 2H+ ----> H2O2
H2O2 + 2e- + 2H+ ----> 2H2O
••O2 + 4H+ + 4e- ----> 2H2O
Eº (V) vs. NHE-0.33+0.89+0.38+2.31
+0.28+1.35
+0.82
O O
HH
2HO•
Homolytic peroxide cleavage
Heterolytic peroxide cleavage: The Fenton Reaction
O O
HH
e-
HO• + HO-
Eº = +0.38 V
Catalyzed by metals like iron and copper
•OH + RH ----> H2O + R•
R• + ••O2 ----> ROO•
RH + ROO• ----> R• + ROOH
O
O
R•
H
O
ORH
O
O
Antioxidants
OO
OHHO
-O O
OO-
OHHO
O O
Ascorbic Acid
OO
OHHO
HO OH
AH2
Ascorbate
AH-
H
R•
OO
OHHO
-O O
- H+
OO
OHHO
O O
Monodehydroascorbate
•A-
OO
OHHO
-O O
R•
OO
OHHO
O O RH
RH
+ H+
Dehydroascorbic acid
A
+H3N COO-
H
O
NH
H S
O
HN COO-
HH
H•R
+H3N COO-
H
O
NH
H S
O
HN COO-
HH
+H3N COO-
O
HN
S
O
NH
COO-
HH
H
H
+H3N COO-
H
O
NH
H S
O
HN COO-
HH
+H3N COO-
O
HN
S
O
NH
COO-
HH
H
H
GSH
GSSG
GS•
InitiationX2 ----> 2X•X• + RH ----> XH + R•PropagationR• + ••O2 ----> ROO•ROO• + RH ----> ROOH + R•TerminationR• + ROO• ----> ROOR R• + R• ----> R2
ROO• + ROO• ----> ROOOOR ----> O2 + ROOR
Free Radical Chain Reactions
X = •OH, •O2-, ••O2
If R = lipid The E/C couple
TerminationR• + EH ----> RH + E•ROO• + EH ----> ROOH + E•RecoveryAH- + E• ----> A•- + EHA•- + E• ----> A + EH
A + NADPH ----> AH- + NADP+
1/6Glucose + NADP+ ----> 1/3CO2 + NADPH
DHAR
DHAR = dehydroascorbate reductasePPP = pentose phosphate pathway
PPP orPhotosynthesis
If R = soluble, C or GSH
TerminationR• + AH- ----> RH + A•-
ROO• + AH- ----> ROOH + A•-
2A•- + H+ ----> AH- + ARecoveryA + NADPH ----> AH- + NADP+
1/6Glucose + NADP+ ----> 1/3CO2 + NADPH
TerminationR• + GSH ----> RH + GS•ROO• + GSH ----> ROOH + GS•2GS• ----> GSSGRecoveryGSSG + NADPH + H+ ----> 2GSH + NADP+
1/6Glucose + NADP+ ----> 1/3CO2 + NADPH
Peroxide reduction
O O
HH
2HO-
H-
Eº = +1.35 V Can be used to extracthydrides from substrates
Oxygen reduction
Eº = +0.82 V Can be used to extracthydrides from substrates
O O
H-
H+
O O
H
H
H-
H+
2H2O
Acetyl-CoA
Some Bacteria/Plants
CO2 fixation
Fungi/plants
Extant ways of fixing CO2
Reductive TCA cycle
Calvin Cycle
Acetyl-CoA Synthase
Reversing the TCA Cycle
Pyruvate
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G <<< 0
∆G <<< 0
∆G <<< 0
∆G <<< 0
How do you reverse KGDH?
Ketoglutarate synthase
2-oxoglutarate:ferredoxin oxidoreductase
Photosynthetic bacteria
Anaerobic bacteria
What about isocitrate dehydrogenase?
This step can be made reversible if you use a different source of electrons. Use NADPH instead of NADH.
Citrate lyase
C
CH2
C
CH2
COO-
COO-HO
EN
-O P
O
O-
O ADP
N P
O
O-
O-E
O-O
C
CH2
C
CH2
COO-
COO-O
OCoAS
H
B
C
CH2
C
CH2
COO-
COO-O
O2-O3PO
H
CoA S
H
B
C
CH2
COO-
COO-
O H3C C
O
SCoA
Pyruvate synthase
Acetyl-CoA + CO2 ---> pyruvate
Pyruvate:ferredoxin oxidoreductase
Photosynthetic bacteria
Anaerobic bacteria
Furdui, C. et al. J. Biol. Chem. 2000;275:28494-28499
Other bacteria
The Calvin cycle.
3CO2 -----> GAP
9 ATP and 6 NADPH
3C53C1
6C3
6C3
1C3
C6
C3+C3
C3+C4
C6+C3
C5
C4
C7+C3
C7
C5
Most important enzyme is Ribulose-5-phosphate carboxylase (Rubisco)
C
2-O3PO O
CH2OPO32-
OHH
E S
H
B
CS O
CH2OPO32-
OHH
E
N
C
O
NH2
HH
CH O
CH2OPO32-
OHH
N+
C
O
NH2
Transketolase: catalyzes the transfer of C2 units
Aldolase: catalyzes the condensation of C3 ketoses with aldoses
C3 + C3 ---> C6C3 + C6 ---> C4 + C5
C3 + C4 ---> C7C3 + C7 ---> C5 + C5
Overal reaction = 5C3 ---> 3C5
1 GAP molecule is made from 3CO2
3CO2 + 9ATP + 6NADPH ---> GAP + 9ADP + 8Pi + 6NADP+
GAP is converted to glucose by gluconeogenesis
C3 + C3 = C6
Aldolase
Reverse of the step in glycolysis
C
C
H O
CH2OPO32-
OHH
CH2OPO32-
C O
CH OH
H
B
CH2OPO32-
C O-
C OH
H
H A
CH2OPO32-
C O
CHO H
C OH
C
H
OHH
CH2OPO32-
C3 + C6 = C4 + C5
Transketolase
CH2OH
C O
CHO H
C OH
C
H
OHH
CH2OPO32-
S
C-
N+X
Y
RCH2OH
C O-
CO H
C OH
C
H
OHH
CH2OPO32-
S
C
N+X
Y
R
H
B
CH2OH
C O-
S
C
NX
Y
R
H
C
O
C OHH
C OHH
CH2OH
CH2OH
C O-
S
C
NX
Y
R
H
C
O
C OH
CH2OH
H
HA
CH2OH
C O-
S
C
N+X
Y
R
C OH
CH2OH
H
CHO H
CH2OH
C O
C OH
CH2OH
H
CHO H
S
C-
N+X
Y
R
Acetyl-CoA synthase
The Wood-Ljungdahl Pathway
2 CO2 ----> Acetyl-CoA
WesternBranch
EasternBranch
CH3, CH2OH, and CHO transfer
CorrinoidCH3 transfer
N
HN
H N
H
W4+S S
S S
OHSeE
C
O
O
W5+S S
S S
OH
SeE
C
-O
O
H-
W4+S S
S S
OHSeE
C
O
H O-
C
O
H O-
-O P
O
O-
O ADP
C
O
H O P
O
O-
O-
B N
HN
H N
C
O
H
Formate dehydrogenase
Can also use Mo
H- from NADPH
N
HN
H N
C
O
H
BN
HN
NHC
OH
HA
N
HN
N+HC
H-
N
HN
NH2CH-
HA
N
HN
HNCH3
Co+NN
N N
HA
Co+NN
N N
CH3
Nin+1S S
SSFe
SFe
FeS
Fe
OC
O
Nin+3S S
SSFe
SFe
FeS
Fe
C
O OHH A
Nin+3S S
SSFe
SFe
FeS
Fe
-CO+
Nin+1S S
SSFe
SFe
FeS
Fe
C
+O
2 e-
H A
CODH: carbon monoxide dehydrogenase
Acetyl-CoA synthase
Co3+NN
N N
CH3
Fe
S
S
S
FeFe
Fe
SS
Nin+
-C
O+
L S
SNi+
N
NCys
Cys
Cys
Cys
Cys
Cys
Nin+S
SNi+
N
N
L
+[S4Fe4]
C-
O+
Co+NN
N N
Nin+S
SNi2+
N
N
L
2+[S4Fe4]
C-
O+
CH3
Nin+S
SNi2+
N
N
L
2+[S4Fe4]
C-
O
CH3
H
SCoA
B
Nin+S
SNi+
N
N
L
+[S4Fe4]
H3CC
S
O
CoA
Assimilating Acetyl-CoA:The glyoxalate cycle
S C
O
CH2CoA
H
B-
S C
O-
CH2CoA S C
O
C-CoA
H
H
S C
O
C-CoA
H
H
-OOC
C
O
H2C COO-
H A -OOC
CH2
OH
CH2
-OOCC
O
SCoA
H
O
H
B-
-OOC
CH2
OH
CH2
-OOCCOO-
Citrate
AcetylCoA
Citrate Synthase
Oxaloacetate
Claisen condensation
Ligase
Aconitase: lyase
C
CH2
C
C
H
CH
O-O
OH
O
O-
B
C
CH2
C
CH
COO-
C
O-O
O
O-
O-
H Mg2+
COO-H
A
C
CH2
C C
O-O
O
O-
H
H
Isocitrate lyase
CoA
S
O
CH2
H
-OO
O
H
B
H+
H
HO
O
S
CH2
COO-
CoA
-O
H
H+
B
CHO
H
COO-
H2C COO-
Malate synthase
COO-
CO H
CH2
H
C
O O-
B
N+
C
O
NH2
R
COO-
CO
CH2
C
O O-
N
C
O
NH2
R
Malate Dehydrogenase
Acetyl-CoA
Some Bacteria/Plants
CO2 fixation
Fungi/plants
Pyruvate
AA’s
AA’s
AA’s,Acetyl-CoA
AA’s
AA’s
FA’s, AA’s
Malate
Pyruvate
Malate dehydrogenase/cytosolic
Oxaloacetate
Glycolytic intermediates
Pyruvate carboxylase
PEP Carboxykinase
∆G ~ 0
∆G <<< 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G ~ 0
∆G <<< 0
∆G <<< 0
Pyruvate Carboxylase
Fructose-1,6-bisphosphate + H2O --->fructose-6-phosphate + Pi
∆G’º = -16.3 kJ/mol
Glucose-6-phosphate + H2O --->glucose + Pi
∆G’º = -13.8 kJ/mol
OH
OH
H
OH
H
OHH
OH
H2C
H
H
H2C
H
CH2OPO32-
OH H
H OH
O
O P
O
O-
O-
O P
O
O-
O-
H
O H
B
HA
H
O H
B
HA
OH
OH
H
OH
H
OHH
OH
H2C
H
OH
H
H2C
H
CH2OPO32-
OH H
H OH
O
OH