Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathway

Control of fluxMetabolic flux = amount of metabolites going through a pathwayper unit time

Flux through glycolysis varies by >100-fold in muscle dependingon the need for ATP

Maintaining homeostasisLevels of the glycolytic intermediates hardly change despite largechanges in flux

What determines flux and homeostasis? THERMODYNAMICS

Koelle, lec15, p18

Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathwayPictorial analogy: water represents flux of metabolites, amount ofwater in flask represents amount of a particular intermediate, pipesbetween flasks are enzymes, vertical drop represents decrease infree energy

ΔG˚ = height difference between flask bottomsΔG = height difference between water levels

Koelle, lec15, p21

Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathway

Control of fluxWhat limits flux through glycolysis?

A. Supply of accessible glucoseAnalogy: If less water is input than the system can carry, theamount of input will dictate the amount of flux

B. Capacity of glycolytic enzymes to process glcAnalogy: Nonequilibrium rxns are shown as skinny pipes that canlimit flow through the system

How regulate activity of nonequilibrium enzymes?• Allosteric effectors• Modifications (phosphorylation)• Altering amount of protein

Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathwayMaintaining homeostasisA. Requires coordinately regulating all nonequilibrium rxnsAnalogy: narrowing just one skinny pipe causes water to back upin the system, narrowing all three does not

B. Intermediates must remain at a constant levelGlycolytic intermediates also involved in “feeding” otherpathways, altering levels interferes with control of other pathways

Koelle, lec15, p24

Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathwayMaintaining homeostasisGlycolytic intermediates are fed in from other pathways

Koelle, lec15, p25

Glycolysis - Enzyme mechanisms4. Regulating a metabolic pathwayMaintaining homeostasisC. Accumulating certain metabolites can have severe consequences

Glycolysis - Regulation3 non-equilibrium reactions that limit fluxAll three must be coordinately regulated to control flux and maintainhomeostasis

Koelle, lec16, p2

Glycolysis - Regulation5. Regulating glycolysisA. Regulation by cellular energy chargeB. Regulation by hormoneC. Regulation to coordinate glycolysis with other pathways

Glycolysis - Regulation5. Regulating glycolysis

A. Regulation by cellular energy chargeFlux of glycolysis increases when [ATP] is needed

ATP, ADP, and AMP are interconverted

adenylate cyclase

ATP + AMP ⇔ 2 ADP

ATP and AMP allosterically regulate phosphofructokinase-1High physiological [ATP] ~ completely blocks PFK-1Inhibition relieved by [AMP][AMP]/[ATP] controls PFK-1 activity

Energy charge affects the Km of PFK-1 for its substrate (F6P)

Glycolysis - Regulation5. Regulating glycolysis

A. Regulation by cellular energy chargeEnergy charge affects the Km of PFK-1 for F6P

Koelle, lec16, p5

PFK-1 is an allosteric enzyme, allosteric effectors bind remote fromactive site and shift equilibrium between high and low affinityconformations

Glycolysis - Regulation5. Regulating glycolysis

A. Regulation by cellular energy chargeWhat about homeostasis?Hexokinase and pyruvate kinase are regulated to match PFK-1

Hexokinase is feedback inhibited by G6PPyruvate kinase is feed-forward activated by F1,6-BP

Koelle, lec16, p6

Glycolysis - Regulation5. Regulating glycolysis

A. Regulation by cellular energy chargeHexokinase versus Glucokinase (isozymes)HK - in most cells

- has a high affinity for glc (Km = 0.1 mM)- allosterically inhibited by its product, G6P

GK - in liver- affinity for glc (Km = 10 mM, blood [glc] = 5 mM)- efficient glc transporters maintain [glc] in liver at 5 mM- enzyme regulated by level of blood glc- after meals blood glc high, excess blood glc moved to liver- inhibited by F6P

Glycolysis - Regulation5. Regulating glycolysis

A. Regulation by cellular energy chargeWhy use PFK as the control point?

Koelle, lec16, p8

PFK is the first “committed step” - first nonequilibrium enzyme not alsoin other pathways

Glycolysis - Regulation5. Regulating glycolysis

B. Regulation by hormone (glucagon)Glucagon -

Glucagon inhibits PFK-2PFK-2 is an allosteric activator of PFK-1 and it synthesizes F-2,6-BPWhen glucagon is around, it binds its receptor on cell surface, activatesa protein kinase that phosphorylates and inactivates PFK-2

Glycolysis - Regulation5. Regulating glycolysis

C. Regulation to coordinate glycolysis with other pathways

Koelle, lec16, p10

Glycolysis - RegulationSummary

Koelle, lec16, p11

Glycolysis - RegulationWhat about NAD+? Why don’t we run out of it?

Koelle, lec16, p12