Protein regulation 3: Serine Proteases

Question 1

Criteria for an efficient enzyme? (With regards to constants, variables, Kd etc.)

Answer 1

Large Vmax
Km small (not overly small, compromise due to k2 influence in various equations)
k2&raquo_space; k-1 (productive collisions)

Question 2

Example of efficient and not(as)-efficient enzymes and their roles

Answer 2

Acetyl cholinesterase - efficient

Complement proteases (killing cascade of the immune system) - sacrifice efficiency for improved specificity

Question 3

What is the transition state?

Answer 3

Transition state (ES#) is the peak energetic state ‘mountain top’ during a reaction -> very short lifetime (picoseconds) ; least stable possible structure

Question 4

Describe a transition state inhibitor

Answer 4

Molecule designed to mimic the configuration of the transition state

-> make good inhibitors, prevent enzyme function

Question 5

Key features of serine proteases?

Answer 5

Catalytic triad - catalysis

Oxyanion hole - stabilising transition state

Substrate binding site - binding to substrate (B-sheet interactions)

Specificity pocket - specificity

Question 6

Approaches to investigating the mechanism and function of an enzyme - use chymotrypsin as an example

Answer 6

Covalent modification (of residues)
Steady-state kinetics
Transient-state kinetics
X-ray crystallography
nuclear magnetic resonance (nmr) spectroscopy
Site-directed mutagenesis (protein engineering)

Question 7

Why are esters often used as substrates in serine protease assays

(possibly come back to this)

Answer 7

Structure mimics natural (Amide) substrate
Product has yellow colour

Use in steady state kinetics

(possibly come back to this)

Question 8

What are ‘burst kinetics’?

Answer 8

Small amount of time (millisecond scale) before steady state kinetics (pre steady state) where reaction progression is non linear (hyperbolic curve)

Detected by Chromophore yellow A410 in Chymotrypsin experiment

Happens due to multiple steps of reaction in enzyme catalysis (fast release of first product, slow release of second product)

Question 9

What is a zymogen?

Answer 9

Inactive substance which is converted into an enzyme when activated by another enzyme

Question 10

Why cant chymotrypsin digest itself?

Answer 10

Synthesised as a zymogen (Chymotripsinogen)

Activated after secretion by partial proteolysis

Chymotripsinogen still has catalytic triad, BUT

No oxy-anion hole - cannot accommodate transition state