Regulation of Glycolysis/GluconeogenesisCitric Acid Cycle / TCA cycle / Kreb’s cycle

Electron Transport Chain (Oxidative Phosphorylation)

3 major points of regulationof glycolysis

Hexokinase

Phosphofructokinase-1

Pyruvate kinase

-32.9

-24.5

-26.4

Each regulatory reactionis held far from equilibrium (large – G)

Each regulatory step in glycolysisis coordinately regulated withgluconeogenesis Substrate Cycle

*

*

Each regulatory step in glycolysis is coordinately regulated with gluconeogenesis

Futile/Substrate cycle:

ATP

ADP

Pi

H2O

Sum: ATP + H20 ADP + Pi

*

If allowed to occur at the same time – utilization of ATP without useful metabolic work being done Cycles provide important means of regulation

GlycolysisGluconeogenesis

Hexokinase

Muscle

Liver

Muscle – High affinity for glucose

Usually saturated and working at maximal rate

Inhibited by glucose 6-phosphate (product inhibition)

Liver – much lower affinity (higher Km)for glucose.

Allows liver to process high levels of glucose

Not inhibited by glucose 6-phosphate

Coordinate Regulation of PFK-1 and FBPase-1 (regulation by energy state of cell)

PFK-1 – inhibited by signals of adequate energy supplies

ATP Citrate

Activated by signals of low energy suppliesADPAMP

FBPase-1 –Inhibited by signals of low energy supplies

AMP

* Commitment step of glucose into glycolysis

Hormonal regulation of glycolysis and gluconeogenesis mediated by fructose 2,6 bisphosphate

F26BP activates PFK-1 (binds to allosteric site and increases its affinity for its substrate (F6-phosphate)

F26BP inactivates FBPase 1 (decreasesits affinity for its substrate (F1,6 BP))

Where does F2,6 Bisphosphate come from?

Hormonal regulation of PFK-2 / FBPase-1

1 bifunctional NZ with 2 separate activities regulated by insulin and glucagon

**Regulation of NZ is by phosphorylation**

Formation of F26BP Breakdown of F26BP

Glucagon stimulates phosphorylation of PFK-2/FBPase 2-Activation of FBPase-2 activity (phosphatase)-Reduction of F26BP- Glycolysis Gluconeogenesis

Insulin stimulates dephosphorylation of PFK-2/FBPase 2-Activation of PFK2 activity (Kinase) -Increase in F26BP - Glycolysis Gluconeogenesis

Enzymes of glycogen metabolism are regulated by allostericand hormonal mechanisms

Glycogen PhosphorylaseGlycogen Synthase

Activation G6P AMP

Inhibition ADP, Pi ATP, G6P, Glucose

Signals of high energy supply (G6P, ATP) stimulate glycogen synthesis andinhibit glycogen breakdown.

Signals of low energy supply (ADP, AMP) stimulate glycogen breakdownand inhibit glycogen synthesis

Allosteric Regulation

Hormonal Regulation of glycogen synthesis and breakdown – Covalent Modification

Glucagon

Activation of PKA (via cAMP)

P of phosphorylase kinase

P of Glycogen phosphorylaseAnd

P of Glycogen synthase

Stimulation of glyc breakdown

Inhibition of glyc synthesis

(A)

(IA)

Glycogen breakdownInhibition of Glycogen

breakdown

Activation of phosphoprotein phosphatase -1

DeP of Glycogen phosphorylase Phosphorylase kinase

(A) (IA)

(IA)

1) Glucose enters hepatocytes through transporter

2) Synthesis of glycolytic enzymes

Fed State - Insulin

3) inactivation of GSK3 and activation of PP1

Activate glycogen synthase and inactivate glycogen phosphorylase Glycolysis Glycogen synthase ↓Glycogen breakdown

Fasting State - glucagon Activation of PKA through cAMP

1) Activates phosphorylase kinase glycogen phosphorylase 2) Inactivates glycogen synthase

3) Phosphorylates PFK-2/FBPase-2 in F2,6BP 4) Inactivates pyruvate kinase

↓Glycolysis ↓Glycogen synthase Glycogen breakdown

Glycolysis

Glucose Pyruvate

Transition/Prep Phase

NADH FADH2

C1 C2 C3 C4ATP

synthase

ATP

CitricAcid Cycle

Electron Transport

Overview of Steps

Acetyl - CoA

Cytoplasm

Mitochondria

Pyruvate

2 C acetyl group combines with 4 C oxaloacetate to yield 6 C citrate

Citrate (C6)

Isocitrate (C6)

-ketoglutarate (C5)

Succinyl co-A (C4)

Succinate (C4)

Fumarate (C4)

Malate

Oxaloacetate (C4)

C02

CoASHC02

NADH

GTP / ATP

CoASHFADH2

NADH

2 Acetyl-CoA

CoASH

Substrate level phosphorylation

1 Glucose

2 pyruvates

NADH

Pyruvate Dehydrogenase

1-

2- Carbons lost in pathway as C02 -These Cs are not from acetyl-coA

3- Oxaloacetate consumed in the 1st step is regenerated in the last.

4- Energy produced is transferred as energy-rich electrons to NAD+ to yield NADH or to FAD+ to yield FADH2

5- 2 rounds of Cycle yields: 4 CO2, 6 NADH, 2 FADH2, and 2 ATP

6- CAC is amphibolic – has a role in oxidation of carbohydrates and provides precursors for other pathways (ex – AA metabolism)

Citric Acid Cycle

Ca2

Ca2

Ca2

Regulation of Citric Acid Cycle

1- Pyruvate Dehydrogenase (irreversible) Product/Feedback inhibition Covalent Modification (Phosphorylation = inactive)

2- Isocitrate Dehydrogenase 4- Citrate synthase

NADH Citrate

NADH and Acetyl CoA compete with NAD+ and CoA for binding sites – competetive feedback inhibition

Product/FeedbackInhibition

***

* All function far from equilibrium (- ΔG)

3- a-Ketoglutarate Dehydrogenase

A- Substrate Availability (oxaloacetate;acetyl-CoA)

B- Product Inhibition

C- Competitive Feedback Inhibition

Regulated enzymes of CAC

1- PD

2- ID

3-KD

4-CS

Electron Transport Chain

Matrix

Intermembrane space

NADH NAD+

MembraneSoluble

Coenzyme Q

FADFeS

Cyt. C

½ O2 + 2H+

H20

4H+ 4H+ 2H+

Complex I

Complex II

ComplexIV

FADH2FAD+

FMNFeS

Succinate Fumarate

Cyt’sFeS

Complex III

Cyt’sCu

1- Electrons from NADH and FADH2 are passed through redox centers of 4 membrane bound complexes and 2 membrane soluble electron shuttles

2-During electron transfer, protons are translocated from the matrix into the intermembrane space (Complex 1, Complex III, and Complex IV)

3- Final reduction is of molecular O2

4- Electrons from NADH yield 2.5 ATP; Electrons from FADH2 yield 1.5 ATP

Proton Motive Force - Chemiosmotic theory

H+ H+ H+

++++++ ++++++

--------- ---------

Low pH

High pH H+

l lll lV V

ATP

Intermembrane space

Outer membrane

Inner membrane

Storage of energy as a proton/voltage gradient across a membrane

1- In ETC, the transport of H+ from low [H+] to high [H+] requires energy (ENDERGONIC)

2- Discharge of proton is EXERGONIC Free energy of discharge of proton gradient is harnessed by ATP synthase

Oxidative phosphorylation

F1

Fo

02 C

on

sum

pti

on

Time

DNP

Time

DNP

AT

P S

ynth

esis

Time

DNP

No drug

Inhibitor addeduncoupler

added

Electron transport and ATP synthesis are “coupled”ATP synthesis requires discharge of proton gradient:

Proton gradient cannot be discharged without synthesis of ATP:Proton gradient is established by electron transporting complexes.

Electrons flowing through ETC; O2 is being consumed;ATP is being synthesized Inhibitor of ETC

Stops flow of electrons;No proton pumping; No proton motive force;No longer consuming O2;No longer making ATP

Uncoupler of ETCDestroys proton gradient; No stored energy for ATP;DOES NOT stop electron flow;Oxygen still being consumed