Enzyme Regulation and Inhibition Notes

Enzyme activity can be regulated by:
- Controlling enzyme activity directly
- Controlling enzyme availability

Phosphorylation is a common covalent modification.
The terminal phosphate group from ATP is attached to the hydroxyl groups of Serine (Ser), Threonine (Thr), or Tyrosine (Tyr).
This process is catalyzed by enzymes called kinases.
Most covalent modifications are reversible.
Example: Phosphate removal by enzymes called phosphatases.
Phosphorylation is a highly effective way of regulating the activities of target proteins.
Residues that can get phosphorylated are Serine, Threonine, Tyrosine.

The phosphate group alters the charge of the protein.
- Introducing new electrostatic interactions within the protein, affecting other charged amino acids.
- Leading to conformational changes (shifts in alpha-helices, beta-sheets, unfolding, or refolding).
- Creating new binding sites for other proteins.
- Activating or deactivating specific structural regions around the active site.

Example: Activation of the insulin receptor by phosphorylation.
The activation loop, when unphosphorylated, is inactive and blocks the substrate-binding site.
Phosphorylation of the activation loop leads to its movement, opening up the substrate-binding site for the target protein.

Inactive (unphosphorylated) tyrosine kinase domain needs to be phosphorylated to become active.
Active (triply phosphorylated) form.
Triply phosphorylated activation loop moves dramatically, making room for the target protein in the substrate-binding site.

Glycogen phosphorylase catalyzes the breakdown of glycogen.
Phosphorylase $a$ : phosphorylated (active) form of the enzyme, with a phosphoryl group on Serine 14.
Phosphorylase $b$ : the dephosphorylated form (less active).
Catalyzes bond cleavage.
This process is rate-limiting in breaking down glycogen.
In muscle, the product is used for ATP synthesis.
In the liver, the product is converted to free glucose.

Isoenzymes (or isozymes) are enzymes encoded by different genes but catalyze the same reaction.
They display different regulatory properties.
Isozymes may be expressed in a tissue-specific or developmentally-specific pattern.
Example: Glycogen phosphorylase isozymes expressed in muscle, brain, and liver.
Isozymes allow fine-tuning of metabolism to meet the needs of a given tissue or developmental stage.

Dimer of 842 residue subunits.
Larger N-terminal domain includes:
- Glycogen binding subdomain
- Interface domain
- Allosteric effector site
- Ser-14 phosphorylation site
The active site is at the center of the subunit.
Regulated by allosteric interactions and phosphorylation/dephosphorylation.
Structures of phosphorylase $a$ and $b$ are similar

T state: surface loop blocks substrate access.
R state: Serine 14 phosphorylation reorients surface loop allowing substrate access.
The phosphate recognition site is fully formed only in the R state: Arg 569.
The addition of substrates to glycogen triggers a T to R allosteric shift.

Phosphorylase needs to be active during muscle contraction.
During muscle contraction, ATP is converted into AMP, signaling phosphorylase to degrade glycogen.
AMP (allosteric effector) binds to a nucleotide-binding site and stabilizes the conformation of phosphorylase $b$ in the R state.
ATP (allosteric inhibitor) competes with AMP and favors the T-state; G6P also favors T-state.

Phosphorylase $a$ exhibits the most responsive T-to-R transition (not the $b$ form).
Binding of glucose (allosteric inhibitor) shifts the $a$ form to the T state from R state, inactivating it.

Blood glucose level increases after a meal; there is no need to break down glycogen into glucose.
Blood glucose level decreases, shifting the equilibrium to the R state to break down glycogen into glucose.
Unlike in muscle, liver phosphorylase is insensitive to regulation by AMP (recall isozyme definition).