Glycolysis

• Glucose → pyruvate (+ ATP, NADH)

• Preparatory phase + Payoff phase

• Enzymes– Highly regulated (eg. PFK-1 inhibited by ATP)– Form multi-enzyme complexes

• Pass products/substrates along: efficiency

Overall balance sheet

Glucose + 2NAD+ + 2ADP + 2Pi →

2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

Fermentation pathwaysAlternate fate of pyruvate

• “Fermentation”: carbohydrate metabolism that generates ATP but doesn’t change oxidation state (no O2 used, no net change in NAD+/NADH)

• Fermentation of pyruvate to lactate– Cells with no mitochondria

(erythrocytes) – anerobic conditions– Regeneration of 2 NAD+ to

sustain operation of glycolysis

– No net change in oxidation state (glucose vs lactate)

• Lactate is recycled to glucose (post-exercise)

Fermentation pathways

• Fermentation of pyruvate to EtOH– Yeast and

microorganisms – No net oxidation

(glucose to ethanol)

– EtOH and CO2 generated

Aerobic respiration of glucose (etc)

• Glycolysis: – Start with glucose (6 carbon)– Generate some ATP, some NADH, pyruvate (2 x 3 carbon)

• TCA cycle– Start with pyruvate– Generate acetate– Generate CO2 and reduced NADH and FADH2

• Electron transport – Start with NADH/FADH2

– Generate electrochemical H+ gradient• Oxidative phosphorylation

– Start with H+ gradient and O2 (and ADP + Pi)– Generate ATP and H2O

Aerobic respiration

• Stage 1:– Acetyl CoA production

• Some ATP and reduced electron carriers (NADH)• Glycolysis (for glucose), pre-TCA

• Stage 2:– Acetyl CoA oxidation

• Some ATP, lots of reduced e- carriers (NADH/FADH2)• TCA cycle/Krebs cycle/Citric acid cycle

• Stage 3:– Electron transfer and oxidative phosphorylation

• Generate and use H+ electrochemical gradient• Use of reduced e- to generate ATP

Fate of pyruvate under aerobic conditions: TCA cycle (Ch. 16)

• Oxidation of pyruvate in ‘pre-TCA cycle’ – Generation of acetyl CoA (2

carbons)

– CO2

– NADH

• Acetyl CoA → TCA cycle– Generation of ATP, NADH

Pre-TCA cycle

• Pyruvate acetyl CoA– Via ‘pyruvate dehydrogenase complex’

• 3 enzymes

• 5 coenzymes

– ~irreversible– 3 steps

• Decarboxylation

• Oxidation

• Transfer of acetyl groups to CoA

• Mitochondria– Transport of pyruvate

Pre-TCA cycle• Coenzymes involved (vitamins)– Catalytic role– Thiamin pyrophosphate (TPP)

• Thiamin• decarboxylation

– Lipoic acid• 2 thiols disulfide formation• E- carrier and acyl carrier

– FAD• Riboflavin• e- carrier

– Stoichiometric role– CoA

• Pantothenic acid• Thioester formation acyl carrier

– NAD+

• Niacin• e- carrier

Pre-TCA cycle

• Enzymes involved pyruvate dehydrogenase complex– multiprotein complex– Pyruvate dehydrogenase (24) (E1)

• Bound TPP

– Dihydrolipoyl transacetylase (60) (E2)• Bound lipoic acid

– Dihydrolipoyl dehydrogenase (12) (E3)• Bound FAD

• 2 regulatory proteins– Kinase and phosphatase

• Step 1: – Catalyzed by

pyruvate dehydrogenase

• Decarboxylation using TPP

• C1 is released

• C2, C3 attached to TPP as hydroxyethyl

Pre-TCA cycle

Pre-TCA• Step 2

– Hydroxyethyl TPP is oxidized to form acetyl linked-lipoamide– Lipoamide (S-S) is reduced in process– Catalyzed by pyruvate dehydrogenase (E1)

• Step 3– Acetyl group is transferred to CoA– Oxidation energy (step 2) drives formation of thioester (acetyl

CoA)– Catalyzed by dihydrolipoyl transacetylase (E2)

• Step 4– Dihydrolipoamide is oxidized/regenerated to lipoamide– 2 e- transfer to FAD, then to NAD+– Catalyzed by dihydrolipoyl dehydrogenase (E3)

Overall….

• Pyruvate acetyl CoA– Via ‘pyruvate dehydrogenase

complex’– 4 step process

• Decarboxylation of pyruvate and link to TPP

• Oxidation of hydroxyethyl TPP and reduction/acetylation of lipoamide

• Transfer of acetyl group to CoA

• Oxidation of lipoamide via FAD (and e- transfer to NAD+)

Overall….

• Pyruvate acetyl CoA– Via ‘pyruvate dehydrogenase

complex’– 4 step process

• Decarboxylation of pyruvate and link to TPP

• Oxidation of hydroxyethyl TPP and reduction/acetylation of lipoamide

• Transfer of acetyl group to CoA

• Oxidation of lipoamide via FAD (and e- transfer to NAD+)

Pre-TCA

• Substrate channeling– Multi enzyme complex rxn rate

• Facilitated by E2 – ‘swinging’ lipoamide– accept e- and acetyl from

E1 and transfer to E3

• Pathology: mutations in complex/thiamin deficiency

Regulation of pre-TCA• PDH complex

– Inhibited by • Acetyl CoA, ATP, NADH, fatty

acids

– Activated by • CoA, AMP, NAD+

– Phosphorylation• Serine in E1 phosphorylated

by kinase– Inactive E1– Kinase activated by ATP,

NADH, acetyl CoA…

• Regulatory phosphatase hydrolyzes the phosphoryl

– Activates E1– Ca2+ and insulin stimulate

TCA cycle• Aerobic process

– “Generates” energy– Occurs in mitochondria– 8 step process

• 4 are oxidations• Energy ‘conserved’ in

formation of NADH and FADH2

– Regenerated via oxidative phosphorylation

– Acetyl group → 2 CO2

• Not the C from the acetyl group

– Oxaloacetate required in ‘catalytic’ amounts

– Some intermediates• Other biological purposes

TCA cycle• Step 1: condensation

of oxaloacetate with acetyl CoA citrate

• Via citrate synthase– Conformational change

upon binding– Oxaloacetate binds 1st

• Conf change to create acetyl CoA site

• Citrate synthase– Conformational changes

upon binding of oxaloacetate

unboundbound

TCA cycle• Mechanism of citrate

synthase• 2 His and 1 Asp• 2 reactions

– 1st rxn (condensation)• 2 steps• Highly unfavorable because

of low oxaloacetate

– 2nd rxn (hydrolysis)• Highly favorable because of

thioester cleavage• Drives 1st rxn forward

• CoA is recycled back to the pre TCA cycle