Biochem Assessment 5

pH changes can…

inhibit enzyme function

Why does pH change enzyme function?

Enzymes have an optimal pH

change in pH —> protonation states

changes structure: H-bonds, ionic/salt bridges

changes in functions: H-bonds, charges, interactions for mechanism

Molecular inhibitor

Molecule that slows/halts enzymatic reaction

Reversible molecular inhibitor

Reaction can continue once inhibitor is gone

Irreversible molecular inhibitor

Binds/destroys functional groups on enzyme; Cannot be restored

Chymotrypsin is a…

digestive enzyme

How is chymotrypsin regulated through protein maturation?

1. Produced as single chain, inactive

2. Small intestine → trypsin cleaves btwn arg15 and ile16

3. Conformational change → New ilr16 N-term flips in & forms salt bridge w/ asp194 → Open active site

4. Autolysis (self cleavage) → 3 chains w/ 5 S-S disulfide bonds

Salt bridge btwn asp194 & N-term of ile16…

opens and stabilizes active site of chymotrypsin

1. makes salt bridge

2. Conformational changes

3. Opens & stabilizes active site

The hydrophobic pocket in chymotrypsin…

helps stabilize the intermediate

How do cells regulate ezymes?

1. protein maturation

2. controlling availability of substrate

3. Controlling availability of enzyme

4. Covalent/posttranslational modification

5. Allostery

Enzymes can be regulated by…

1. Phosphorylation

2. Adanylylation

3. Acetylation

4. Myristoylation

5. Ubiquitination

6. ADP-ribosylation

7. Methylation

Protein kinases (add/remove) phosphates; phosphatases (add/remove) phosphates

add; remove

positive allostery modulator

fascilitates better substrate binding

negative allosteric modulator

locks to substrate binding site (less accessible)

y = m x + b

1/V₀ = (k_m/V_max * 1/[S]) + 1/V_max

competitive inhibition

Competing with S for active site

Km increased, affinity decreased

k_m/V_max (m) changes

uncompetitive inhibitor

Binds outside active site (interaction with E is constant)

V_max decreases

1/[S] (b) changes

Mixed inhibitor

Binds outside active site

Km can increase or decrease

k_m/V_max (m) AND 1/[S] (b) changes

Noncompetitive inhibitor

Inhibitor binds E and ES with equal affinity

ATCase is a(n)…

allosteric enzyme

How does changing pH cause inhibition in enzymes?

• Disrupting ionic/salt bridges/H-bonds - gain/lose e-

• Change conformation in active site - no longer fits S or catalyst

  • Structural bonds break - Protein unfolds/denatures, E loses function

pH vs Velocity graph: protonated is (decreasing/increasing); deprotonated is (decreasing/increasing)

decreasing; increasing