Lecture 9: Regulation of glycolysis

Why may the rates of metabolic processes vary

In response to changing environment conditions

What 2 ways can this happen

Rapid (seconds/minutes)

Long term (hours/days)

What is the rapid response usually in reaction to

Changes in enzyme activity

And the long term?

Changes in gene expression/ protein synthesis of the enzyme

What is the usual blood glucose level in the body

5mM

If blood glucose increases above this, what is triggered and how does this decrease glucose

Glycolysis, where glucose is turned into pyruvate

If blood glucose decreases below 5mM what is triggered

Gluconeogenesis

What are the 3 factors controlling flux through metabolic pathways

Control (enzyme controls whether reaction is inhibited/activated), direction (enzyme changes direction of reaction) and heat (enzyme controls amount of heat generated by reaction to avoid protein damage)

What 4 factors control fluxes through metabolic pathways

Substrate availability

Concentration of enzymes

Allosteric regulation of enzymes

Covalent modification of enzymes

In glycolysis, what is the substrate

Glucose

What are the 3 rate limiting enzymes in glycolysis

Hexokinase, phosphofructokinase and pyruvate kinase

What is enzyme concentration in glycolysis controlled by

Hormones (insulin and glucagon)

What controls allosteric regulation of enzymes in glycolysis

Binding of small molecules to sites other than the active site

What is the enzyme used in the covalent modification of enzymes

Pyruvate kinase

What is the uptake of glucose into a cell regulated by

the GLUT family of transporter proteins

What are the 3 GLUT transporters + where are they located

GLUT 1 - RBCs

GLUT 2 - Liver and pancreatic beta cells

GLUT 4 - Muscle cells and adipocytes

What are the function of GLUT 1 transporters

Controls basal glucose uptake/ how much glucose enters the bood

What are the functions of GLUT 2 transporters

Uptake glucose at a rate proportional to amount of glucose present

Also remove excess glucose from blood

What is the function of GLUT 4 transporters and at are they regulated by

Remove excess glucose from the blood and are regulated by insulin

What are the 2 positions of the GLUT integral membrane proteins

T1 - open to outside of cell

T2 - open to inside of cell

If the blood glucose level is below 5mM, what is the resting state of ion channels, GLUT2 pumps, and insulin vesicles when glucose enters the pancreatic beta cell

Glucose enters cell through GLUT 2 (substrate not THAT available)

The glucose undergoes glycolysis, producing ATP which causes polarisation of cell

K+ channels are open, so K+ leaves cell

Insulin vesicles sit stably in cell

When blood glucose increases above 5mM what then happens

More glucose undergoes glycolysis so more ATP produced

K+ channels are ATP dependent so close

Causes hyperpolarisation which opens VG Ca2+ channels

Influx of Ca2+ causes insulin vesicles to fuse with membrane and release insulin by exocytosis

When blood glucose is below 5mM what happens in muscle cells

Insulin receptor inactive as no insulin has been released

Glut 4 vesicles remain inside cell

But if this raises to above 5mM what happens to these vesicles

Insulin binds to receptors which activates the GLUT 4 vesicles causing them to fuse with the membrane and transport glucose into the cell

Once glucose is transported into the cell what happens to it, using what enzyme

It is converted into glucose-6-phosphate by hexokinase (with Mg+)

Reaction also produces ADP from ATP

Te glucose-5-phosphate is trapped within the cell

What is particular about the rate limiting enzymes of glycolysis

They indicate the irreversible reactions

What reaction does hexokinase catalyse

Glucose + ATP → Glcose-6-phosphate + ADP

What reaction does PFK catalyse

Fructose-6-phosphate + ATP → Fructose-1,6-bisphosphate + ADP

What reaction does pyruvate kinase catalyse

Phosphoenolpyruvate + ADP → pyruvate + ATP

The concentrations of key rate-limiting enzymes are regulated by

Hormones

For example, how does insulin binding affect the 3 main enzymes action

Binding of insulin to receptor activates a transcriptional activator, which uregulates the expression of hexokinase, PFK and pyruvate to reduce glucose concentration (triggers more glycolysis)

What does the presence of glucagon do to these enzymes

Inhibits them

Define allosteric control

Inhibition or activation of an enzyme by a small regulatory molecule that interacts at a site (allosteric) other than the active site which modifies the enzyme activity

Why may allosteric binding inhibit enzyme action

Binding of the regulatory molecule changes the active site conformation so the ligand (substrate) can no longer bind and the reaction can’t be catalysed

What is hexokinase inhibited by (allosteric product inhibition)

Glucose-6-phosphate

Hexokinases are a family of enzymes, there are 4 types and locations, what’s re they

Hexokinase 1 → brain

Hexokinase 2 → Muscle and adipose tissue

Hexokinase 3 → Kidney

Hexokinase 4 → liver

What is special about hexokinase 4/ glucokinase

It is the only hexokinase NOT inhibited by glucose-6-phosphate

What does this mean for glucokinase

It keeps working as long as glucose is abundant, it isn’t inhibited by glycolysis

When glucose isn’t abundant, glucokinase attaches to a regulatory protein and the complex is sequestered (kept separate from cytosol) in the nucleus until glucose rises again

Why is PFK the most important control site in the mammalian glycolysis pathways

It catalyses the first unique step in glycolysis/ acts as a pace enter for the recess

What 2 molecules is PFK 1 allosterically inhibited by

ATP and citrate (so inhibited by the citric acid cycle AND oxidative phosphorylation)

What 2 molecules is PFK 1 allosterically activated by

AMP and Fructose-2,6-bisphosphate

Why is PFK-2 bifunctional

It acts as PFK-2 and FBPase (Fructose-2,6-bisphosphotase) but is a single protein

How does this work - when is each sub-enzyme activated

PFK-2 converts fructose-6-phosphate into fructose-2,6-bisphosphate (more glycolysis so activated in high glucose)

FBPase-2 converts Fructose-2,5-bisphosphate into fructose-6-phosphate (less glycolysis SO activated in low glucose)

What primarily controls PFK in skeletal muscle

ATP and AMP levels (ATP inhibits, AMP activates -i.e. during exercise)

What 2 molecules inhibits pyruvate kinase

ATP and Acetyl CoA

What activates pyruvate kinase

Fructose-1,6-bisphosphate

What does it mean that pyruvate kinase is regulated by a feedforward mechanism

It is activated by a previously made product in glycolysis which lies further upstream, as the enzyme is downstream of the product, instead of the product activating/inhibiting an enzyme that lies further up

How else is pyruvate kinase regulated

Covalent modification/ phosphorylation

Phosphorylation makes pyruvate kinase more active