Transcript Energy Metabolism - Georgia Institute of Technology

Energy Metabolism
• ATP synthesis
– Outline the steps of glycolysis
– Outline the steps of lipolysis
– Citric acid cycle/Electron transport chain
• Control processes
– Explain the contribution of mass action to the rate
of ATP synthesis
– Similarly, allosteric feedback
Phospho-creatine ATP buffer
• Creatine Kinase
– Unique to striated muscle
– Creatine + ATP ADP + phospho-creatine
• Creatine
– 20-40 mM total creatine
– 16-32 mM phospho
– ATP ~ 5-10 mM
Glycolysis
• Convert Glucose to Pyruvate
– Yield 2 ATP + 2 NADH per glucose
– Consume 2 ATP to form 2x glyceraldehyde
phosphate
– Produce 2 ATP + 1 NADH per GAP
• Carefully controlled
– 12 different enzyme-catalyzed steps
– Limited by phosphofructokinase
– Limited by substrate availability
Glycolysis: phosphorylation
• ATP consuming
– Glucose phosphorylation by hexokinase
– Fructose phosphorylation by phosphofructokinase
• Triose phosphate isomerase
Glycolysis: oxidation
• Pyruvate kinase
– Transfer Pi to ADP
– Driven by oxidative
potential of 2’ O
• Summary
– Start C6H12O6
– End 2xC3H3O3
– Added 0xO
– Lost 6xH
– Gained 2xNADH, 2xATP
GAPDH
NADH
ATP
phosphoglycerate kinase
pyruvate kinase
Pyruvate
• Lactic Acid
– Regenerates NAD+
– Redox neutral
• Ethanol
– Regenerates NAD+
– Redox neutral
• Acetyl-CoA
– Pyruvate import to mitocondria
– ~15 more ATP per pyruvate
pyruvate
2-HydroxyethylThiamine diphosphate
S-acetyldihydrolipoyllysine
Acetyl-CoA
Carbohydrate metabolism depends on transport
• H+, pyruvate cotransporter
Major Facilitator Superfamily
Monocarboxylate transporter
Competition between H+
driven transport to
mitochondria and
NADH/H+ driven
conversion to lactate
Cytoplasmic NADH is
also used to generate
mitochondrial FADH2,
coupling transport to
ETC saturation
“glycerol-3P shuttle”
Halestrap & Price 1999
Gluconeogenesis
• During contraction, inefficient glycolysis
wastes glucose
– Many glycolytic enzymes are reversible
• Special enzymes
– Pyruvate carboxylase
– Phosphoenyl pyruvate carboxykinase
• Swap carboxyl group for phosphate
• Generates 3-C phosphoenolpyruvate from OA
– Fructose-1,6-bisphosphatase
• Generates fructose-6-phosphate
Mitochondrial
• Generate 4-C oxaloacetate from 3-C pyruvate
Fatty Acid/b-oxidation Cycle
1x FADH2
1x NADH
Acetyl-CoA
– 3x NADH+
–1xFADH2
• Acyl(n)-CoA + NAD+ + FAD
Acyl(n-2)-CoA + Acetyl-CoA + NADH +FADH2
Carnitine palmitoyltransferase
Fatty acid elongation
Acyl-CoA
Acyl-CoA synthase
FAD
Acyl-CoA
dehydrogenase
Acyl-CoA
FADH2
acetyl-CoA
acyltransferase
Acetyl-CoA
Didehydroacyl-CoA
Acyl-CoA hydrase
CoA-SH
3-hydroxyacyl-CoA
dehydrogenase
Hydroxyacyl-CoA
Oxoacyl-CoA
NADH
NAD+
Reactive oxygen
• FADH2 oxidative stress
– Succinate; saturated FA
– FADH2 + Fe3+  FADH • + H+ + Fe2+
– Fe2+ + H2O2Fe3+ + OH- + OH•
• FADH2 more completely reduces UQ than does NADH
Acyl-CoA
Acyl-CoA
FAD
Acyl-CoA
dehydrogenase
UQ
ETF:QO oxidoreductase
FADH2
Didehydroacyl-CoA
FADH2
FAD
O2
Acyl-CoA
oxidase
UQH2
H2O2
Didehydroacyl-CoA
Free fatty acids from triglycerides
• FFA cleavage from circulating lipoproteins
– Protein/cholesterol carriers: Lipoprotein
• Density inversely correlates with lipid
• Correlates with cholesterol/FA (except HDL)
• VLDL & LDL to IDL
– Lipoprotein lipase (LPL)
– HDL scavenges cholesterol & facilitates IDL breakdown
• Triglycerides are retained in intracellular droplets
– Don’t fit in membrane (no phosphate)
– Not water soluble
Fatty acid metabolism depends on transport
• FAAcyl-CoA Acyl-Carnitine Acyl-CoA
Cytoplasm
Intermembrane
Matrix
Working substrate
Boron & Boulpaep
Mitochondrial Transport
•
•
•
•
•
Carrier protein (FABP)
Long chain acyl-CoA synthetase (LCAS)
Cross outer membrane via porin
Convert to acylcarnitine in intermembrane
Cross inner membrane via
carnitine:acylcarnitine transferase
• Convert back to acyl-CoA in matrix
Mitochondrial Structure
• Principal metabolic engine
• Symbiotic bacteria
– 6k-370kBP genome
– Human: 13 proteins
• Dual membrane
– ie: two bilayers
– Outer membrane highly
permeable
– Inner membrane highly
impermeable
Mitochondrial Matrix
• Highly oxidative environment
• Unique proton gradient
– High pH (8), negative (-180 mV), ~18 kJ/mole
– H+ actively transported out of matrix
– H+ leak back as H+PO4 2-
• Capture gradient energy for ATP synthesis
– H+ ATPase pump
– ADP-ATP antiporter
• Other proton co-transporters
– Pyruvate, citrate
– Glutamate, citruline
Metabolic Substrates
• Sugars
– Metabolized in cytoplasm to pyruvate
– Co-transported to matrix with H+
– Bound to Coenzyme A as Acetyl-CoA
• Fatty acids
– To intermembrane space as Acyl-CoA
– To matrix as Acyl-carnitine
– Metabolized to Acetyl-CoA in matrix
• Proteins
CH3
C=O
COO-
Acetyl Coenzyme A
• Common substrate for oxidative metabolism
• S-linked acetate carrier
The Citric Acid Cycle
Acetyl-Coenzyme A
CoA
These carbons will be
removed
NADH
Oxaloacetate
New carbons
Citrate
Carbon
Oxygen
Malate
Coenzyme A
Isocitrate
NADH +
=
Fumarate
a-Ketoglutarate
FADH2
Succinate
CoA
Succinyl CoA
+ GTP
NADH
CoA
Electron transport
• Couple NADH/FADH2 electrons to H+ export
– Ideally this completes NADH + H+ + ½ O2
NAD+ + H++2e½O2+2 H++ 2e-
NADH DE0=-0.32V
H2O
DE0=0.82V
– Electron leakage
NAD+ +H2O
KEGG pathway
Enzyme Commission (EC) number
•Hierarchical
•Function-centric nomenclature
•Compare
•Gene Ontology (GO) ID
•Entrez RefSeq
•UniProt ID
Metabolite
KEGG http://www.genome.jp/kegg/pathway.html
Cyclic redox reactions
Oxidized
NAD+
FAD
NADH
CoQ/ubiquinone
Cyto-C3+
O2
dihydroubiquinone
Cyto-C2+
H 2O
FADH2
Reduced
You can only have this progressive
redox process if molecular position is
carefully controlled
NAD+  NADH
FAD FADH2
Ubuquinone
Cytochrome C
O2  H2O
E0 = -0.32V
E0 = -0.22V
E0 = 0.10V
E0 = 0.22V
E0 = 0.82V
Proton ATPase/Complex V
• ATP driven proton pump
– “Reversible”
– Couples H+ gradient to ATP synthesis
Fatty acid/carbohydrate oxidation
• Oxygen
– CnH2n + 3/2 n O2  n CO2+ n H2O
– CnH2nOn +n O2 n CO2 + n H2O
– Respiratory Quotient CO2/O2
• 0.67 Fatty acids
• 1.00 Carbohydrates
• Adenine electron transporters
– 6-C glucose6 NADH + 2 FADH2 (3:1)
– 16-C FA  32 NADH + 16 FADH2 (2:1)
• Redox chemistry differs for FA/CHO
Muscle substrate utilization
• Rest: fatty acids
• Active: glycolysis
• Recovery:
– Pyruvate oxidation
– Gluconeogenesis
Role of mass action in flux control
• Diffusion
– J = D ∂f/∂x (greater flux down a steeper gradient)
– ∂f/ ∂t= ∂J/∂x
• Kinetics
– d[P]/dt = k[S] (1st order)
– d[P]/dt = Vmax [S]/(Km + [S]) (Michaelis-Menten)
– d[P]/dt = k [S1][S2] (2nd order)
Mass action in glycolysis
• Diffusion
– Substrate consumption increases gradient
– Increased gradient accelerates mass flow
• Kinetics
– G+ATPG6p d[G6p]dt = k1[G][ATP]≈k[G]
– G6pF6p
d[F6p]/dt = k2[G6p]
– F6p+ATPF1,6p <etc>
– F1,6pG3p+DAp
– DApG3p
Mitochondrial substrate dependence
• More ADPfaster ATP
– Discharge proton gradient
– Lower ETC resitsance
• More NADfaster
– Faster NADH
– Greater ETC input
Wu &al 2007
Role of allosteric regulation
• Allosteric
– Binding to other-than-active site changes enzyme
Allosteric ADP binding site
kinetics
– Vmax or kM
• Many metabolic enzymes are regulated by
downstream products
– Phosphofructokinase
• Citrate inhibits
• ADP activates
– Gylcogen synthase
Active site
PDB:3PFK
G6P regulation of GS
• Allosteric conformational change
Without G6P
Less active
With G6P
More active
Baskaran et al. 2010
Role of post-translational regulation
• Chemical modification of enzymes alters
activity
– Phosphorylation
– Ribosylation, acylation, SUMOylation, etc
– Integrative response to complex conditions
• Insulin
– Insulin  IRPI3KGLUT4 translocation
glucose uptake
–
PI3KPKB--|GSK--|GS
Phospho-regulation of glycogen
• PKA
• PKB
+GP via phosphorylase
kinase
-GS
-PP1 via G-subunit
+GS via GSK
+PP1 via G-subunit
•PP1
+GS
-GP
Activates
Inhibits
PK
PKA
GP
PP1-G
PP1
GS
GP
Glycogen
Synthesis
PP1
PP1-G
GS
GSK3
PKB
AMP kinase
• Allosterically activated by AMP
– Adenylate kinase: 2 ADP  AMP + ATP
– ADP levels insensitive to energy state
PFKglycolysis
--|GSGlyconeogenesis
--|ACCMalonyl CoA--|CPTFA oxidation
--|ACClipogenesis
TSC2--|mTOR…protein synthesis
--|HMGCoAcholesterol synthesis
Summary
• Sources of ATP
– Creatine
– Gylcolysis: GG3p2OPA
– Lipolysis: acyl-CoAoxoacyl-CoA
– Citric Acid Cycle/Electron Transport Chain
• AcCoACitrate...Oxaloacetate
• Rate control by
– Mass action
– Allosteric feedback
– Hormonal control