BCM. 26 - GLYCOLYSIS

Kinase

An enzyme that adds a phosphate group from ATP onto another molecule. i.e. they catalyse X + ATP → phospho-X + ADP.

The phosphorylated product will usually have a name like “phospho-X/ “X-phosphate”/ “X-bisphosphate”/ X-diphosphate”,

-Hexokinase

Difference between diphosphate and bisphosphate

Diphosphate = two phosphate groups chained off the same atom

Bisphosphate = two separate phosphate groups attached at different positions

A protein kinase is

A kinase that specifically adds a phosphate group from ATP onto another protein.

This target protein is often an enzyme or a transcription factor, and the phosphorylation results in the enzyme’s or TF’s activity increasing (or decreasing).

The phosphate is almost always added to one of the three alcoholic amino acids (serine, threonine or tyrosine), and the resulting phosphorylated protein is called a phosphoprotein.

-Protein-kinase A, which phosphorylates many proteins in the cell

-Glycogen-phosphorylase kinase, which phosphorylates the enzyme glycogen-phosphorylase.

Phosphorylases

Add phosphate groups to a chemical from inorganic phosphate (not from ATP), i.e. they catalyse X + Pi → phospho-X.

- Glycogen phosphorylase, which reacts glycogen with inorganic phosphate to make glucose-phosphate, and in-so-doing reduces the length of the glycogen polymer (glycogenn + Pi → glycogenn–1 + glucose-phosphate). Glycogen phosphorylase is itself regulated by phosphorylation.

Phosphatase

The action of a kinase or a phosphorylase can be undone by a phosphatase, which is an enzyme that removes a phosphate from another molecule by hydrolysis,

i.e. they catalyse phospho-X + H2O → X + Pi.

- Fructose-bisphosphatase, which removes one of the phosphates groups from fructose-1,6-bisphosphate.

- Phosphoprotein phosphatases are phosphatases that remove phosphate groups from phosphorylated proteins; thereby undoing the action of a protein kinase.

Phosphorylation

The metabolic process of introducing a phosphate group into an organic molecule.

allosteric regulation

The binding of a regulatory molecule to a protein at one site that affects the function of the protein at a different site.

Allosterically regulated enzymes are usually ...

Each catalytic subunit can exist in either a...

The R and T forms exist in ...

When substrate binds to any one relaxed subunit,...

This results in...

This is called....

...multimeric, with more than one catalytically active subunit.

Tense (T) form, which has low affinity for substrate; or a relaxed (R) form, which has high affinity.

Equilibrium when the enzyme is not bound to substrate.

This information is communicated through structural changes to some or all of the other subunits.

that other subunit converting to the relaxed form, thus making it easier for a second substrate molecule to bind.

co-operativity.

Not only do some substrates react when they bind to the enzymes, it also make the enzyme's other active sites more active. This is called .

It's positive because ...

and its homotropic ('same-changing') because...

positive homotropic modulation.

the binding of substrate makes the enzyme more active,

the substrate helps other molecules of the same type to bind.

Allosterically regulated enzymes show..

sigmoidal kinetics:

•The sigmoidal (allosteric) enzyme has a much sharper response between [S] = 1 … 2

Than MM enzymes

Le Chatelier's Principle

States that if a stress is applied to a system at equilibrium, the system shifts in the direction that relieves the stress.

Allosteric inhibitors only bind to

tense forms

Allosteric activators only bind to

relaxed form

A ligand is a ..

That includes ....

Ligands that act ...

small molecule that sticks to a larger one

substrates, but also inhibitors and activators.

as allosteric modulators can either be positive or negative in their effects.

An allosteric activator is a ...

Modulation can also be ...

positive heterotropic modulator: one molecule is facilitating the binding of a different kind of molecule.

modulator that increases the affinity of the enzyme for some molecule.

heterotropic ('other-changing').

one molecule is facilitating the binding of a different kind of molecule.

Why does positive heterotrophic modulators work?

If the concentration of the AMP-bound relaxed form [R-AMP] increases, then then the equilibrium will favour conversion of enzymes in the T form to the R form. Therefore [T] falls, and [R] increases. This is an application of Le Chatelier’s principle.

An allosteric inhibitor is

How does it work?

a modulator that decreases the affinity of the enzyme for some molecule.

In the example shown on the left of this slide, the ATP is acting as a negative heterotropic modulator: when the ATP binds to the enzyme, it locks the subunits into the tense state, preventing the binding of substrate: one molecule (ATP) is inhibiting the binding of a different kind of molecule (the substrate).

3 stages of glycolysis

Prepatory

Cleavage

Pay off x 2

Glycolysis occurs in the

It is a ... reaction

Only involves...

cytosol

oxidation

SLP

Overall equation for glycolysis

Glucose + 2ADP + 2Pi + 2NAD > 2 pyruvate + 2ATP + 2NADH

•Glucose from

•Glycogen stored

•Starch stored in

food

in liver

amyloplasts

Glycogen phosphorylase is activated by a .... in the...in response to ..

kinase cascade

liver

glucagon

glycogen phosphorylase activation

alternative?

1) Protein kinase A activated by G-DCR/ CAMP pathway

2) PKA activates phosphorylase kinase via phosphorylation

3) PK phosphorylates and activates glycogen phosphorylase

4) GP turns glycogen > glucose phosphate

Another way may be to use hexokinase to turn glucose to the glucose-P.

Draw glycolysis steps

Per glucose:

-2 ATP in

-4 ATP out

-2 NADH out

= net production 2 ATP

Only 1 ATP is needed if the substrate is

glycogen rather than glucose, but note that the original synthesis of glycogen from glucose requires ATP. Breaking the glycosidic bond in glycogen to make glucose-P expends the energy put in to making the glycogen, and this amount of energy is similar to that required to directly phosphorylate glucose to glucose-phosphate.

"Enzymes that are regulated" often

–Have negative ∆G

–Act in forwards/ backwards pairs e.g. PFK and PBP

Most are allosterically regulated.

The forwards/backwards reactions have to be...

because ...

different, since if ∆G is negative for the forward reaction, the backwards reaction cannot be spontaneous. Hence the 'backwards' version of the reaction must be a different reaction with a different Keq. For PFK/FBP, the sugars are the same, but the other reactants differ.

PFK and FBP reaction

catalyse different reactions in glycolysis and gluconeogenesis.

not a true reverse reaction as it doesnt generate ATP

Bacterial PFK is a ...

multimeric enzyme showing cooperative binding for fructose-P.

PFK is

allosterically regulated by many metabolites.

PEP uses

It is

ADP / AMP is the

negative feedback with PFK to control the process.

allosteric inhibitor

allosteric activator

PFK shows

sigmoidal kinetics w.r.t. [Frc-P], and is modulated allosterically by ATP and AMP:

Cleavage involves...

aldolase and triose-P isomerase

1 hexose-P → 2 triose-P

•Triose phosphate =

-Dihydroxyacetone phosphate

-Glyceraldehyde phosphate

The later steps generate

1 NADH and 2 ATP per GAP:

enol pyruvate is unstable so

this drives the PK reaction rightwards

Define SLP

Substrate-level phosphorylation is a metabolic reaction that results in the formation of ATP or GTP by the direct transfer of a phosphoryl (PO3) group to ADP or GDP from another phosphorylated compound.

Driven by instability of enol-pyruvate Vs Keto-pyruvate.

Pyruvate kinase is regulated by

positive feed-forward from Frc-BP:

•Ensures FBP and PK work in concert

The...equation can be used to model the binding of ligands showing cooperativity.

The gradient is

Hill

cooperativity coefficient.

Values of n in Hills equation

Note that if n is 1, and L is the substrate then the kinetics are just Michaelis-Menten-like, with a hyperbolic shape, and a Kd that simply is the KM.

If n is larger than 1, then the ligands are cooperating: see that the kinetics are now sigmoidal, with a much sharper increase to θ approaching 1: the ‘upstroke’ is nearly vertical here – no activity…no activity…suddenly all the activity).

If n is less than 1, then the ligands are interfering: you get slightly more activity than for n=1 at very low [L], but then the increase is even slower than for a Michaelis-Menten-like kinetics

Ancestral vs Diverged mammalian PK

Ancestral:

- Sensitive to PEP inhibitor

- Active site shows cooperative binding for Frc-P in the ancestral enzyme (homotropic positive modulation by a phosphosugar)

Diverged:

- Sensitive to inhibition by citrate and AMP/ATP

- Lower subunit cannot phosphorylate

- Fructose-2,6-bisphosphate is an extremely potent activator of PFK1 made by PK2 when [fructose-P] is too high

- One of the ‘active sites’ has been repurposed as a allosteric activator binding site (heterotropic positive modulation by a phosphosugar)