Regulation of Metabolic Pathways

• Systems must respond to conditions

• Homeostasis is not equilibrium

• Dynamic Steady State

– Flux - Rate of metabolic flow of material through pathways

• Many ways to regulate – for example

– At the protein level (e.g. allosteric control)

– At the gene level

– At transcription or translation

• There are different time scales for regulation

– < sec, seconds, hours, days

– Based on situation that requires response

• Maintaining ATP concentration is critical– Energy needed to sustain cellular processes– Typical cell

• [ATP] 5 mM• ATP-using enzymes KM range 0.1 – 1 mM• Significant [ATP] drop would cause many

reactions to decrease

• Cells are sensitive to ratiosATP/ADP(or AMP)NADH/NAD+

NADPH/NADP+

ATP + glucose ADP + glucose 6-phosphate

• AMP is a very sensitive indicator – small changes make a big difference percentage-wise (normal conc. <0.1 mM)

]][[

]6][[ln0

gluATP

PGADPRTGG

-Fast response (sec or less) – usually allosteric control (faster response than synthesis or degradation of enzyme)

-Covalent modification (also fast)most common: phosphorylation/dephosphorylation

-Slower response (sec to hours) –exterior effects such as hormones, growth factors

Overall regulatory networks will:1. maximize efficience of energy source

utilization by preventing futile cycles.2. partition metabolites between alternative

pathways (Ex: glycolysis and PPP).3. use the best energy source for the immediate

needs of the cell.4. shut down biosynthetic pathways when their

products accumulate.

Vocabulary:Metabolic regulation – maintains homeostasis at

the molecular level (e.g. hold concentrations of metabolites constant)

Metabolic control – changes flux through a metabolic pathway

Coordinated Regulation of Glycolysis & Gluconeogenesis

Futile (substrate) cycles are to be avoided

cycles that recycle metabolites at the expense of ATP

Glycolysis Regulation

• When ATP is needed, glycolysis is activated

• When ATP levels are sufficient, glycolysis activity decreases

Control points1. Hexokinase 2. PFK-1 3. Pyruvate kinase

1. Hexokinase• Hexokinase reaction is metabolically

irreversible• G6P (product) levels increase when glycolysis

is inhibited at sites further along in the pathway

Recall there are 4 isozymes• G6P inhibits hexokinase isozymes I, II and III • Glucokinase (hexokinase IV) forms G6P in

the liver (for glycogen synthesis) when glucose is abundant (activity is modulated by fructose phosphates and a regulatory protein)

• Isozymes I,II and II have similar KM (important in muscle)– Normally at saturation

• Hexokinase IV has much higher KM (important in liver)– Important when blood glucose is high

• Glucose enters mammalian cells by passive transport down a concentration gradient from blood to cells

• GLUT is a family of six passive hexose transporters

• Glucose uptake into skeletal and heart muscle and adipocytes by GLUT 4 is stimulated by insulin

• Other GLUT transporters mediate glucose transport in and out of cells in the absence of insulin

• GLUT2 is transporter for hepatocytes• Quick equilibrium of [glucose] with blood

glucose in both cytosol and nucleus• Regulator protein – inside the nucleus

– Binds Hexokinase IV and inhibits it– Protein has regulatory site

• Competition between glucose and fructose 6-phosphate

– Glucose stimulates release of hexokinase IV into cytoplasm

– Fructose 6-phosphate inhibits this process• Hexokinase IV not affected by glucose 6-

phosphate as the other isozymes are

Addition of a regulatory protein raises apparent KM for glucose (inhibits hexokinase IV)

Glucose 6-Phosphate Has a Pivotal Metabolic Role in Liver

2. Regulation of Phosphofructokinase-1

• Important - this step commits glucose to glycolysis

• PFK-1 has several regulatory sites

• ATP is a substrate and an allosteric inhibitor of PFK-1 (note that it’s an end-product of the pathway)

• AMP allosterically activates PFK-1 by relieving the ATP inhibition (ADP is also an activator in mammalian systems)

• Changes in AMP and ADP concentrations can control the flux through PFK-1

•AMP relieves ATP inhibition of PFK-1

• Elevated levels of citrate (indicate ample substrates for citric acid cycle) also inhibit PFK-1

• Most important allosteric regulator is fructose 2,6-bisphosphate (later in the chapter)

3. Regulation of Pyruvate Kinase (PK)

• At least 3 PK isozymes exist in vertebrates

• Differ in distribution and modulators

• Inhibited by high ATP, Acetyl-CoA, long-chain fatty acids (energy in good supply)

Liver form – low blood sugar glucagon increased cAMP cAMP-dependent protein kinase PK inactivation (is reversed by protein phosphatase)

• Muscle form – epinephrine→increased cAMP → activates glycogen breakdown and glycolysis

• PK is allosterically activated by Fructose 1,6 BP

• PK inhibited by accumulation of alanine

Regulation of Gluconeogenesis

• Fate of pyruvate

•Go on to citric acid cycle – requires conversion to Acetyl Co-A by the pyruvate dehydrogenase complex

•Gluconeogenesis – first step is conversion to oxaloacetate by pyruvate carboxylase

• Acetyl Co-A accumulation

• inhibits pyruvate dehydrogenase

• activates pyruvate carboxylase

Coordinated regulation of PFK-1 and FBPase-1 (1) Phosphofructokinase-1 (PFK-1) (glycolysis)(2) Fructose 1,6-bisphosphatase FBPase-1 (gluconeogenesis)

• Modulating one enzyme in a substrate cycle will alter the flux through the two opposing pathways

• Two coordinating modulators•AMP•Fructose 2,6-bisphosphate

• Inhibiting PFK-1 stimulates gluconeogenesis

• Inhibiting the phosphatase stimulates glycolysis

• AMP concentration coordinates regulation• stimulates glycolysis• Inhibits gluconeogenesis

• In the liver, the most important coordinating modulator is fructose 2,6-bisphophate (F2,6BP)

• It is formed from F6P by the enzyme phosphofructokinase-2 (PFK-2)

• It is broken down by the same enzyme, but at a different catalytic site in the enzyme – it’s a bifunctional protein

-It is called fructose 2,6-bisphosphatase (FBPase-2) for this activity

- Balance of PFK-2 to FBPase-2 activity controlled by

-Glucagon

-Insulin

• F2,6BP stimulates glycolysis

• F2,6BP inhibits gluconeogenesis

Effects of Glucagon and Insulin

The Pasteur Effect

• Under anaerobic conditions the conversion of glucose to pyruvate is much higher than under aerobic conditions (yeast cells produce more ethanol and muscle cells accumulate lactate)

• The Pasteur Effect is the slowing of glycolysis in the presence of oxygen

• More ATP is produced under aerobic conditions than under anaerobic conditions, therefore less glucose is consumed aerobically

Regulation of Glycogen Metabolism

• Muscle glycogen is fuel for muscle contraction

• Liver glycogen is mostly converted to glucose for bloodstream transport to other tissues

• Both mobilization and synthesis of glycogen are regulated by hormones and allosterically

• Insulin, glucagon and epinephrine regulate mammalian glycogen metabolism (hormones)

• Ca2+ and [AMP]/[ATP] (muscle glycogen phosphorylase)

• [glucose] (liver glycogen phosphorylase)

• [glucose 6-phosphate] (glycogen synthase)

• Hormones

•Insulin is produced by -cells of the pancreas (high levels are associated with the fed state)

-increases glucose transport into muscle, adipose tissue via GLUT 4 transporter-stimulates glycogen synthesis in the liver

• Glucagon is Secreted by the cells of the

pancreas in response to low blood glucose

(elevated glucagon is associated with the

fasted state)

-Stimulates glycogen degradation to

restore blood glucose to steady-state

levels

-Only liver cells are rich in glucagon

receptors

• Epinephrine (adrenaline) Released from the

adrenal glands in response to sudden energy

requirement (“fight or flight”)

- Stimulates the breakdown of glycogen to

G1P (which is converted to G6P)

-Increased G6P levels increase both the

rate of glycolysis in muscle and glucose

release to the bloodstream from the liver

Reciprocal Regulation of GlycogenPhosphorylase and Glycogen Synthase

• Glycogen phosphorylase (GP) and glycogen synthase (GS) control glycogen metabolism in liver and muscle cells

• GP and GS are reciprocally regulated both covalently and allosterically (when one is active the other is inactive)

• Covalent regulation by phosphorylation (-P) and dephosphorylation (-OH)

COVALENT MODIFICATION (Hormonal control)

Active form “a” Inactive form “b” Glycogen phosphorylase -P -OHGlycogen synthase -OH -P

Allosteric regulation of GP and GS

GP a (active form) - inhibited by GlucoseGP (muscle)- stimulated by Ca2+ and high [AMP]

GS b (inactive form) - activated by Glucose 6-Phosphate

• Hormones initiate enzyme cascades

•Catalyst activates a catalyst activates a catalyst, etc.

• When blood glucose is low: epinephrine and glucagon activate protein kinase A

• Glycogenolysis is increased (more blood glucose)

• Glycogen synthesis is decreased