Protein Regulation: Covalent Modification and Proteolysis

Covalent modification involves making or breaking chemical bonds to switch a protein to its active or inactive form, changing its tertiary structure and acting as an on/off switch.

Understand covalent modification, focusing on phosphorylation.
Learn about proteolysis, where cleaving parts of proteins activates or inactivates them.

Phosphorylation:
- Covalent attachment of a phosphate group to amino acids (serine, threonine, or tyrosine) within the protein.
Acylation:
- Attachment of aldehydes or ketones to the protein.
- Important for gene transcription by controlling histones that package DNA.
Attachment of Lipid Groups:
- Localizes proteins at the cell membrane.
- Example: Signaling proteins near the membrane to generate IP3 from lipids.
Ubiquitination:
- Ubiquitin (a protein) is covalently attached to a protein, signaling its degradation.
- Targets the protein to proteasomes for degradation via proteolysis.

Involves the transfer of the terminal phosphate of ATP to serine, threonine, or tyrosine residues in the protein.
A bond is formed between the hydroxyl group in serine/threonine/tyrosine and the phosphate of ATP.
Enzymes:
- Kinases: Transfer the phosphate group onto the protein.
- Phosphatases: Remove the phosphate group.
Two irreversible, opposite counter-reactions.
The phosphorylation state of a protein is determined by the balance between kinase and phosphatase activity.
Phosphorylation is related to the energy status of the cell since ATP is used.

Adding a phosphate group changes the protein's capacity to interact with other amino acids.
Phosphate groups introduce negative charges and hydrogen bonding capacity.
This alters amino acid interactions, leading to conformational changes that activate or deactivate the protein.

Catalyzes the breakdown of glycogen.
Exists in two states: inactive and active.
Phosphorylation by a kinase activates the enzyme; dephosphorylation by a phosphatase inactivates it.
The hydroxyl-containing amino acid phosphorylated is a serine residue.
Introduction of negative charges and hydrogen bond acceptor capability causes a conformational change, unmasking the active site.
Inactive form of the protein has a hidden blue amino acid; phosphorylation changes the structure to reveal the active site.

Enzymes (kinases and phosphatases) can modify many target molecules.
If target molecules are also kinases/phosphatases, there can be amplification.
A small amount of activated enzyme can activate many of a second enzyme, and so on.
Phosphorylation cascades (e.g., MAP kinases in cell signaling).

Proteolysis involves cleaving peptide bonds.
It is typically a one-way switch.
A protein is switched on by proteolysis and switched off by irreversible inhibition.
Phosphorylation has two competing, opposite reactions (phosphorylation/dephosphorylation).

Trypsinogen (inactive) is cleaved by enteropeptidase to form trypsin (active).
Trypsin then cleaves other zymogens like chymotrypsinogen, procarboxypeptidase, and proelastase.
Inactive zymogens are converted to active enzymes by proteolysis by a protease.

Cleavage of trypsinogen to trypsin by enteropeptidase causes a conformational change.
A new terminus is created, which tucks into the protein structure, activating the protein.

Requires an inhibitor.
Example: Trypsin inhibitor binds to the active site of trypsin (competitive inhibitor).
The inhibitor is not a substrate and is not proteolyzed.
Inhibitors are important to prevent trypsin from being active all the time.
Over-activation of trypsin can lead to damage of the pancreas (acute pancreatitis).

Cascade of proteases.
Factor XII is activated to factor XIIa, which activates factor XI to factor XIa.
Factor IX is activated to factor IXa, which joins with factor VIII to form factor Xa.
Factor Xa cleaves prothrombin (factor II) to form thrombin (factor IIa).
Thrombin cleaves fibrinogen to form soluble fibrin.
Fibrin aggregates to form an insoluble mesh.
This fibrin mesh captures cells and plugs holes in blood vessels.

Thrombin cleaves fibrinogen to fibrin.
Fibrinopeptides are cleaved out of fibrinogen, creating a new N-terminus.
This N-terminus binds to the C-terminus domain, producing a lattice network of fibrin.

Cascades of enzymes working on enzymes can lead to amplification.
A small change at the top of the cascade can lead to a large change at the bottom.

Covalent modification and proteolytic cleavage are key mechanisms for regulating protein activity.
Both processes can amplify signals through enzyme cascades.

The general overview of phosphorylation is as follows:
$Protein + ATP \xrightarrow{Kinase} Protein-PO_4 + ADP$
The reverse reaction, dephosphorylation, is shown below:
$Protein-PO<em>4 + H</em>2O \xrightarrow{Phosphatase} Protein + P_i$