Lecture 20 (Protein Regulation, Allostery, Phosphorylation & Degradation)

What are apparent Vmax/Km effects of reversible inhibitors?

• Competitive: Vmax same, Km ↑

• Uncompetitive: Vmax ↓, Km ↓

• Mixed: Vmax ↓, Km ↑ or ↓

• Noncompetitive: Vmax ↓, Km same

What are the major ways proteins are regulated?

• Allosteric regulation/feedback inhibition

• Covalent modification/phosphorylation

• Cellular compartmentalization

• Zymogen activation

• Protein synthesis/gene regulation

• Protein degradation

What are zymogens?

Inactive precursor forms of enzymes

What is a good enzyme candidate for regulation?

Start of pathway enzyme with a strongly favored forward reaction

What is the allosteric modulator?

May be substrate in which the allosteric site and the active site are the same.

• Example of homotrophic allosteric modulation

What are allosteric enzymes?

Usually multimeric enzymes regulated by ligand binding at sites affecting activity

What is homotropic allostery?

Substrate acts as allosteric modulator

What is heterotropic allostery?

Non-substrate modulator binds a separate regulatory site

What do allosteric activators do?

• Enhance catalytic activity

• Decrease Km/K0.5 and/or increase Vmax

What do allosteric inhibitors do?

• Decrease catalytic acitivity

• Increase Km/K0.5 and/or decrease Vmax

What is cooperativity in allosteric enzymes?

Binding one ligand changes activity/affinity at other sites

What is positive vs negative cooperativity?

• Positive enhances activity/affinity

• Negative decreases activity/affinity

What is feedback inhibition?

Late pathway product binds an allosteric/regulatory site on an early enzyme and inhibits it

Is feedback inhibition reversible?

Yes; dissociation of the modulator reverses the conformational change

What is positive regulation?

Product from one pathway stimulates an early enzyme in another pathway

How are allosteric and active sites linked?

Binding at one site affects binding/activity at the other site

How can high substrate oppose feedback inhibition?

Substrate stabilizes active form, reducing affinity for allosteric inhibitor

What is catabolism?

Breakdown of nutrients to energy-depleted products; energy stored in ATP/NADH

What is anabolism?

Synthesis of complex molecules from simple ones; requires free energy

What indicates a high-energy cell state?

High ATP, acetyl-CoA, and citrate

What indicates a low-energy cell state?

High ADP and AMP

How do energy signals affect catabolism?

ATP inhibits; ADP/AMP activate

How do energy signals affect anabolism?

ATP activates; ADP/AMP inhibit

What residues are phosphorylated for regulation?

Ser, Thr, and Tyr

What does protein phosphorylation do?

Reversibly changes conformation or enables binding/signaling

How common is phosphorylation in eukaryotic cells?

About 1/3 of proteins are phosphorylated at a time

What enzymes phosphorylate proteins?

Kinases and phosphorylases

How do kinases vs phosphorylases differ?

Kinases transfer phosphate from ATP; phosphorylases transfer phosphate from Pi

What enzymes remove phosphate groups?

Protein phosphatases

Can phosphorylation activate or inhibit enzymes?

Yes; it can switch enzymes on or off

How is pyruvate dehydrogenase regulated by phosphorylation?

Phosphorylation inactivates PDH; dephosphorylation activates it

What stimulates PDH phosphorylation/inactivation?

Acetyl-CoA and NADH

What stimulates PDH dephosphorylation/activation?

ADP and Ca2+

What inhibits PDH phosphorylation?

High NAD+ and pyruvate

How does compartmentalization regulate enzymes?

Signal sequences target enzymes to specific compartments where they act

How is trypsinogen activated?

Enteropeptidase cleaves an N-terminal peptide, creating active trypsin

How are proteases prevented from unwanted activity?

Protease inhibitors bind tightly to active sites

What is the proteasome?

Eukaryotic machinery that degrades proteins

What is proteasome structure?

20S core plus two 19S caps

How are proteins targeted to proteasome?

Polyubiquitin tags

What is ubiquitin?

76-AA protein covalently attached to Lys side chains via isopeptide bonds

What enzymes mediate ubiquitination?

E1 activates ubiquitin, E2 carries it, E3 ligase attaches it to target

How can degradation signals be revealed?

Phosphorylation, conformational change, complex dissociation, or protease cleavage

What is the N-end rule?

N-terminal amino acid identity can determine protein half-life/degradation rate

How can new N-termini trigger degradation?

Protease cleavage reveals an N-terminal residue that acts as a degradation signal