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

NADHATP

pyruvate kinase

GAPDH

phosphoglycerate kinase

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

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”

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

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

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

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

• FAAcyl-CoA Acyl-Carnitine Acyl-CoACytoplasm 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

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

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

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

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

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 glucose6 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 ∂/∂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)

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

• More ADPfaster ATP– Discharge proton gradient– Lower ETC resitsance

• More NADfaster– Faster NADH– Greater ETC input

Mitochondrial substrate dependence

Wu &al 2007

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

G6P regulation of GS• Allosteric conformational change

Without G6PLess active

With G6PMore 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 IRPI3KGLUT4 translocation

glucose uptake– PI3KPKB--|GSK--|GS

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

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

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