L22: Proteolytice Enzymes

Question 1

Acid catalysis

Answer 1

Strong acid dissociates readily-> give free proton -> protonates carbonyl oxygen -> withdrawing e from carbonyl carbon -> more susceptible to nucleophilic attack by water

Bond formation between water oxygen and carbonyl carbon: associated with transition state

Question 2

General acid catalysis

Answer 2

Weak acid (H-A) which protonates carbonyl oxygen as water oxygen nucleophilically attacks carbonyl oxygen and assists process by stabilising developing -ve charge on carbonyl oxygen (C=O bond is broken)

Transition state: carbonyl carbon-hydroxyl oxygen bond formed

Question 3

Metal ion catalysis

Answer 3

Analogous to acid catalysis

Question 4

General base catalysis

Answer 4

Weak base assists nucleophilic attack of water oxygen kn carbonyl carbon by at same time removing proton from water molecule

Question 5

Proteases

Answer 5

Enzymes that catalyse hydrolytic cleavage of peptide bonds in proteins and peptides

1. Nucleophilic attack on carbonyl carbon (can be directly by water or nucleophile in enzyme active site like AA side chain or cofactor)
2. Development of -ve on carbonyl oxygen
3. Protonation of amide nitrogen and breaking of C-N bind

Some are specific in that the peptide bond to be hydrolysed must be adjacent to particular type of AA

Question 6

Chymotrypsin catalytic triad

Answer 6

His57, asp102, ser195

His57- general base -> general acid catalysis -> general base catalysis-> general acid catalysis

Hydroxyl oxygen of Ser195 nucleophilically attacks carbonyl carbon of peptide bond -> hydrolysed.
Assisted by side chain nitrogens of His57: act as general base catalyst -> remove proton from hydroxyl group of Ser195 during nucleophilic attack on carbonyl oxygen
As proton transferred to His57, +ve charge develops on imidazole ring (stabilised by proximity of -ve charged carboxylate of Asp102 & HB between it and imidazole proton of His57) -> stabilises tetrahedral transition state
Peptide bond breaks, proton from protonated His57 transferred to leaving amino group of formed C-terminal peptide -> leaves N terminal peptide covalently bound to Ser195 hydroxyl oxygen in acyl-enzyme intermediate
His57 again acts as general base catalyst -> assisting nucleophilic attack of water molecule on carbonyl carbon of acyl-enzyme intermediate.
Acyl bond between N terminal peptide & hydroxyl oxygen of Ser195 is broken -> release N terminal peptide

Question 7

Proteases have similar catalytic triad and function in same way as chymotrypsin

Answer 7

Have different specificities (nature of active site pocket -> binds side chain of AA N-terminal to peptide bond to be broken)

-vely charged carboxylate of Asp189 in binding pocket of trypsin -> electrostatic & HB interactions with +vely charged amine groups of side chains of Arg or Lys in substrate protein

Large hydrophobic pocket in chymotrypsin accomodates bulky aromatic residues, tend to be hydrophobic

Small pocket in elastase -> allow small AA side chains to bind

Question 8

Thiol proteases

Answer 8

Catalytic diad: Cys and His. Operate in similar way as Ser proteases except sulphur of Cys thiol nucleophilically attacks carbonyl carbon of substrate

Question 9

Metalloproteases

Answer 9

Metal ion bound at active site performs metal ion catalysis (stabilising developing -ve charge on carbonyl oxygen of peptide bond as water nucleophilically attacks carbonyl carbon)

In carboxypeptidase A: general base catalysis by Glu270. Tyr248 may play role in proton transfer between hydroxyl group and amino nitrogen

Question 10

Acid or aspartate proteases

Answer 10

Have essential active site aspartate residues

pH profile of the proteases: bell shaped. pH optimum around 3. -> 2 acidic AA residues were involved kn catalysis: one needing to be protonated and one deprotonated

Proposed mechanism for aspartate proteases: 2 active site Asp residues- one initially deprotonated and one is protonated. Deprotonated: acts as general base catalyst. Protonated: acts as general acid catalyst

Question 11

HIV-1 protease

Answer 11

Essential for viral propagation (catalyses proteolytic cleavage of initial polyprotein product of HIV-1 genome into its constituent active proteins)

pH profile suggested similarity with aspartate proteases such as pepsin. Pepsin: active site formed between 3 domains, with 1 Asp coming from each domain. HIV-1: active site formed between 2 subunits of dimer, with 1 Asp coming from each subunit

Knowledge of structure, specificity and mechanism of HIV-1 protease -> drugs designed to bind tightly to enzyme activity site, with hydroxyl group positioned between 2 Asp residues -> strongly inhibit enzyme