energy metabolism atp synthesis – outline the steps of glycolysis – outline the steps of...
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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
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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
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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
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Glycolysis: phosphorylation• ATP consuming
– Glucose phosphorylation by hexokinase– Fructose phosphorylation by phosphofructokinase
• Triose phosphate isomerase
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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
NADHATP
pyruvate kinase
GAPDH
phosphoglycerate kinase
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Pyruvate• Lactic Acid
– Regenerates NAD+– Redox neutral
• Ethanol– Regenerates NAD+– Redox neutral
• Acetyl-CoA– Pyruvate import to mitocondria– ~15 more ATP per pyruvate
pyruvate2-Hydroxyethyl-
Thiamine diphosphate
S-acetyldihydro-lipoyllysine Acetyl-CoA
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Carbohydrate metabolism depends on transport
• H+, pyruvate cotransporter
Halestrap & Price 1999
Major Facilitator SuperfamilyMonocarboxylate 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”
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Gluconeogenesis• During contraction, inefficient glycolysis
wastes glucose– Many glycolytic enzymes are reversible
• Special enzymes– Pyruvate carboxylase
• Generate 4-C oxaloacetate from 3-C pyruvate
– Phosphoenyl pyruvate carboxykinase• Swap carboxyl group for phosphate• Generates 3-C phosphoenolpyruvate from OA
– Fructose-1,6-bisphosphatase• Generates fructose-6-phosphate
Mitochondrial
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Fatty Acid/-oxidation Cycle• Acyl(n)-CoA + NAD+ + FAD
Acyl(n-2)-CoA + Acetyl-CoA + NADH +FADH2
FAD
FADH2
NAD+NADH
CoA-SH
Acyl-CoA dehydrogenase
Acyl-CoA hydrase
3-hydroxyacyl-CoA dehydrogenase
acetyl-CoA acyltransferase
Carnitine palmitoyltransferase
Fatty acid elongation
Acyl-CoA synthase Acyl-CoA
Didehydroacyl-CoA
Hydroxyacyl-CoAOxoacyl-CoA
Acetyl-CoA
Acyl-CoA
1x FADH21x NADHAcetyl-CoA– 3x NADH+–1xFADH2
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Reactive oxygen
Acyl-CoA
Didehydroacyl-CoA
FAD
FADH2
Acyl-CoA dehydrogenase
Acyl-CoA
Didehydroacyl-CoA
O2
H2O2
Acyl-CoA oxidase
UQ
UQH2
• FADH2 oxidative stress– Succinate; saturated FA– FADH2 + Fe3+ FADH • + H+ + Fe2+
– Fe2+ + H2O2Fe3+ + OH- + OH•
• FADH2 more completely reduces UQ than does NADH
FADH2
FAD
ETF:QO oxidoreductase
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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
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Fatty acid metabolism depends on transport
• FAAcyl-CoA Acyl-Carnitine Acyl-CoACytoplasm Intermembrane Matrix Working substrate
Boron & Boulpaep
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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
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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
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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
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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-
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Acetyl Coenzyme A• Common substrate for oxidative metabolism• S-linked acetate carrier
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Oxygen
Coenzyme A
Carbon
Isocitrate
a-Ketoglutarate
Succinyl CoASuccinate
Fumarate
=
Malate
Oxaloacetate
CoA
CoA
CoA
NADH +
NADH+ GTP
FADH2
NADH
The Citric Acid Cycle
Citrate
Acetyl-Coenzyme A
These carbons will be removed
New carbons
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Electron transport• Couple NADH/FADH2 electrons to H+ export
– Ideally this completes
– Electron leakage
NADH + H+ + ½ O2 NAD+ +H2O
NAD+ + H++2e- NADH E0=-0.32V½O2+2 H++ 2e- H2O E0=0.82V
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KEGG pathway
KEGG http://www.genome.jp/kegg/pathway.html
Enzyme Commission (EC) number•Hierarchical•Function-centric nomenclature•Compare
•Gene Ontology (GO) ID•Entrez RefSeq•UniProt ID
Metabolite
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Cyclic redox reactions
Oxidized
Reduced
NADH
FADH2
NAD+
FAD CoQ/ubiquinone
dihydroubiquinone
Cyto-C3+
Cyto-C2+
O2
H2O
NAD+ NADH E0 = -0.32VFAD FADH2 E0 = -0.22VUbuquinone E0 = 0.10VCytochrome C E0 = 0.22VO2 H2O E0 = 0.82V
You can only have this progressive redox process if molecular position is carefully controlled
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Proton ATPase/Complex V• ATP driven proton pump
– “Reversible”– Couples H+ gradient to ATP synthesis
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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 glucose6 NADH + 2 FADH2 (3:1)
– 16-C FA 32 NADH + 16 FADH2 (2:1)
• Redox chemistry differs for FA/CHO
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Muscle substrate utilization• Rest: fatty acids• Active: glycolysis• Recovery:
– Pyruvate oxidation– Gluconeogenesis
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Role of mass action in flux control• Diffusion
– J = D ∂/∂x (greater flux down a steeper gradient)– ∂/ ∂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)
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Mass action in glycolysis• Diffusion
– Substrate consumption increases gradient– Increased gradient accelerates mass flow
• Kinetics– G+ATPG6p d[G6p]dt = k1[G][ATP]≈k[G]
– G6pF6p d[F6p]/dt = k2[G6p]
– F6p+ATPF1,6p <etc>– F1,6pG3p+DAp– DApG3p
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• More ADPfaster ATP– Discharge proton gradient– Lower ETC resitsance
• More NADfaster– Faster NADH– Greater ETC input
Mitochondrial substrate dependence
Wu &al 2007
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Role of allosteric regulation• Allosteric
– Binding to other-than-active site changes enzyme kinetics
– Vmax or kM
• Many metabolic enzymes are regulated by downstream products– Phosphofructokinase
• Citrate inhibits• ADP activates
– Gylcogen synthase
PDB:3PFK
Allosteric ADP binding site
Active site
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G6P regulation of GS• Allosteric conformational change
Without G6PLess active
With G6PMore active
Baskaran et al. 2010
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Role of post-translational regulation• Chemical modification of enzymes alters
activity– Phosphorylation– Ribosylation, acylation, SUMOylation, etc– Integrative response to complex conditions
• Insulin– Insulin IRPI3KGLUT4 translocation
glucose uptake– PI3KPKB--|GSK--|GS
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Phospho-regulation of glycogen
• PKA+GP via phosphorylase
kinase-GS-PP1 via G-subunit
• PKB+GS via GSK+PP1 via G-subunit
•PP1+GS-GP
PKA PKB
PK
PP1-G
GS
PP1-G
GS
GP
PP1 PP1
GSK3
GlycogenSynthesis
GP
Activates
Inhibits
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AMP kinase• Allosterically activated by AMP
– Adenylate kinase: 2 ADP AMP + ATP– ADP levels insensitive to energy state
PFKglycolysis--|GSGlyconeogenesis--|ACCMalonyl CoA--|CPTFA oxidation--|ACClipogenesisTSC2--|mTOR…protein synthesis--|HMGCoAcholesterol synthesis
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Summary• Sources of ATP
– Creatine– Gylcolysis: GG3p2OPA– Lipolysis: acyl-CoAoxoacyl-CoA– Citric Acid Cycle/Electron Transport Chain
• AcCoACitrate...Oxaloacetate
• Rate control by– Mass action– Allosteric feedback– Hormonal control