10 - Enzymes V: Catalytic Mechanisms

Question 1

Multiple substrate reactions

Answer 1

Nearly 2/3 of enzyme reactions have two substrates and two products
S1 + S2 <=> P1 + P2
Each substrate will has its own KM
Problem: Michaelis-Menten model is not valid if 2 substrates can vary in concentration
Solution: Have one substrate fixed at high concentration

Question 2

Multiple substrate reaction kinetics

Answer 2

Pseudo - Michaelis-Menten kinetics:
• Keep concentration of S1 constant and in excess
• Vary the concentration of S2
• Lineweaver-Burk determines apparent KM apparent (S2)
• Repeat process with S2 constant and in excess

Question 3

Kinetics can inform mechanism

Answer 3

S1 + S2 <=> P1 + P2
Different reaction mechanisms are possible:
• Both substrates bind at once i.e. form a ternary complex
- Catalysis occurs after both substrates bind
OR
• Substrates bind and leave sequentially
- Catalysis occurs after first substrate binds
Kinetic analysis can distinguish between these mechanisms

Question 4

Sequential displacement reactions

Answer 4

Reactions where a ternary complex forms

Question 5

Sequential displacement reaction: Ordered mechanism

Answer 5

Substrates bind and leave sequentially

Question 6

Double displacement: ping-pong mechanism

Answer 6

• Characterised by formation of a substituted enzyme intermediate (E’)
• Known as ping-pong as substrates bounce in and out

Question 7

Mechanisms produce distinct kinetics

Answer 7

Michaelis-Menten model can help to identify the reaction mechanism:
• Keep concentration of substrate S2 constant
• Vary the concentration of substrate S1
• Repeat with different concentration of S2
• Lineweaver-Burk plot characteristic of mechanism (cf inhibition)

Question 8

Sequential displacement reactions LB plot

Answer 8

Lineweaver-Burk plot similar to mixed inhibition, but:

• Increasing S2 decreases slope
• Apparent Vmax increases & KM decreases

Question 9

Double displacement reactions LB plot

Answer 9

• Lineweaver-Burk plot similar to uncompetitive inhibition:

• Increasing S2 decreases intercepts
• Apparent Vmax & KM increase

Question 10

Chymotrypsin

Answer 10

• A proteolytic enzyme used for protein digestion in animals
• Cleaves peptide bonds selectively on C-terminal side of large hydrophobic amino acids (Trp, Tyr, Phe, Met)
polypeptide + H2O <=> Peptide 1 + peptide 2
• Double displacement mechanism

Question 11

Mapping the active site

Answer 11

• We can use irreversible inhibitors to identify catalytic residues
• Irreversible inhibitors bind covalently to the enzyme, and tend to react with the most reactive residue
• The most reactive residue tends to be in the active site

Question 12

Irreversible inhibitors

Answer 12

• group-specific reagents
• affinity labels
• suicide inhibitors

Question 13

Group-specific reagents

Answer 13

– React with specific amino acid side chains

e.g. -OH, -SH

Question 14

Affinity labels

Answer 14

– Substrate analogues that bind to active site, like S, but then bind irreversibly and block the active site

Question 15

Suicide inhibitors

Answer 15

– Substrate analogues that are converted into an affinity label by the action of the enzyme. e.g. 5-flurouracil

Question 16

Mapping the active site of chymotrypsin

Answer 16

DIPF = diisopropyl phosphofluoridate, OH group-specific reagent
• Only Serine 195 is modified - most reactive of 27 serine’s in chymotrypsin

Question 17

The chymotrypsin catalytic triad

Answer 17

His57 & Asp102 increase the reactivity of Ser195
• His57 polarises OH group of Ser195 -Acts as a general base catalyst by accepting H+ ion
• Ser195 becomes a highly reactive alkoxide ion - A powerful nucleophile
• Asp102 assists by positioning His57 and countering the positive charge

Question 18

Peptide hydrolysis by chymotrypsin

Answer 18

1. Substrate binds through R1 hydrophobic interactions.
2. Proton abstraction from Ser195 forms alkoxide ion
3. Alkoxide ion attacks the peptide carbonyl group, forming a tetrahedral transition state
4. Proton transferred from His57 to C-terminal NH of the peptide, cleaving the peptide bond, and forming the acyl-enzyme intermediate.
5. Release of the R2-peptide
6. His57 binds a water molecule, and removes a proton
7. The hydroxyl ion attacks the alkyl-enzyme carbonyl group, forming a second tetrahedral transition state
8. Proton is transferred from His57 to Ser195, cleavage of the peptide bond occurs and the R2-peptide is released.
9. Regeneration of active site

Question 19

Evolution of the catalytic triad

Answer 19

• Many proteolytic enzymes use a catalytic triad to achieve cleavage of the stable peptide bond using a similar mechanism.
• Has evolved independently at least 3 times.
• Chymotrypsin and relatives e.g. trypsin, elastase.
• Subtilisin-like proteases – Typified by bacterial subtilisin, but similar structure in other organisms too.
• Wheat Carboxypeptidase II and relatives – Use His and Cys rather than His and Ser.

Question 20

Site-directed mutagenesis

Answer 20

• Catalytic triad in subtilisin: S221, H64 & D32
• Each mutated to Alanine
• Ser & His mutations reduce activity 1 million times.
• Asp mutation 1/20,000
• But Ser/His mutants still 1000 times rate of hydrolysis in free solution

Question 21

Substrate-specificity of serine-proteases

Answer 21

• Chymotrypsin, trypsin and elastase are all serine-proteases.
• Around 40% amino acid sequence identity & almost identical 3D structures.
BUT:
• Chymotrypsin cleaves after amino acids with large hydrophobic side chains (Trp, Tyr, Phe & Met)
• Trypsin cleaves after amino acids with long, positively charged side chains (Arg & Lys)
• Elastase cleaves after amino acids with small side chains (Ala & Ser)