Enzyme catalysis

Question 1

4 mechanisms of catalysis

Answer 1

Nucleophilic catalysis
GABC
Metal ion catalysis
Hydrophobic

Question 2

Chymotrypsin specificity and kinetics

Answer 2

Cleaves C-terminal side of large hydrophobic amino acids
Kinetics observed by p-nitrophenylacetate, which produces p-nitrophenolate (coloured product)
Biphasic kinetics due to fast first step, which forms acyl-enzyme intermediate and slow second step, which regenerates the enzyme

Question 3

Identification of catalytic triad

Answer 3

DIFP is an irreversible inhibitor that modified Ser195 residue - means it’s highly nucleophilic

TPCK mimics a natural substrate - binds to His57

Alanine Scanning -
His57 or Ser195 resulted in 10^6 lower kcat, but Km unaffected.
All 3 residues mutated still had some catalysis - reflects contribution of oxyanion hole.

Question 4

Crystal structure of chymotrypsin

Answer 4

Crystal structure revealed catalytic triad that formed a charge relay - generating highly nucleophilic Ser195
Asp102 - His57 - Ser195

Also revealed oxyanion hole that stabilised oxyanion via H-bonds with N-H of polypeptide backbone

Binding pocket lined with hydrophobic residues - hence prefers large hydrophobic substrates

Question 5

Catalysis of chymotrypsin

Answer 5

Substrate binding - hydrophobic interactions

Ser O- attacks peptide carbonyl, forms tetrahedral oxyanion intermediate

Collapse of oxyanion tetrahedral intermediate, Protonates of amino group by His57, making it a better LG

Incoming H2O is deprotonated by His57, increasing nucleophilicity.

OH- attacks C=O, generates second tetrahedral oxyanion intermediate.

Collapse is facilitated by donation of H+ to regenerate Ser195, reforming the enzyme

Question 6

Carboxypeptidase A mechanism

Answer 6

Zn2+ catalyses direct attack of H2O on the peptide bond - Zn facilitates deprotonation of H2O and makes it more nucleophilic.

Question 7

pH profile of chymotrypsin

Kcat and 1/Km

Answer 7

Kcat increases at high pH (since second step involving OH- as a nucleophile is rate-limiting)

1/Km increases at low pH (since protonation of N-terminal Ile16 is required for salt bridge formation that stabilises binding pocket)

Overall optimum pH is 8

Question 8

Engineering substrate specificity of proteases

Answer 8

Elastase has preference for smaller substrates - as it has a smaller binding pocket lined with bulkier side-chains

G226A mutation in trypsin could alter substrate preference (Arg-X to Lys-X), but made the enzyme less efficient overall

Question 9

ATCase enzyme activity

Answer 9

Converts carbamoyl phosphate and aspartate to N-Carbamoylaspartate
First step of pyrimidine synthesis (UTP and CTP)

Question 10

Determining the structure of ATCase and function of subunits

Answer 10

Disruption of Cys interactions by mercurial reagents removes allosteric properties but not catalytic properties - can be centrifuged to 2 catalytic trimers (with MM kinetics) and 3 regulatory dimers (which bind CTP/ATP)

Reversed by adding excess thiol

Crystallised with PALA - bisubstrate analog, showed each catalytic trimer has 3 active sites

Question 11

Elucidating mechanism of ATCase catalysis

Answer 11

Comparison between PALA-bound and unbound ATCase showed large change in quaternary structure

Lys84 to Arg mutation caused large decrease in Kcat - indicating it is important for catalysis

Question 12

ATCase quarternary structure changes, allosteric regulation, and evidence

Answer 12

Co-operative binding - binding of one substrate promotes T-to-R transition - sigmoidal kinetics

Crystal structures of ATCase with CTP bound - showed that it is in the T-state.
CTP binds to T-state and stabilises it. ATP binds to same site but stabilises R-state

T-to-R transition showed by incorporation of a catalytically inactive catalytic trimer that was fluorescently labelled on a Tyr residue. Fluorescence changed upon binding of substrate to the other catalytic trimer.

Question 13

IgG crystallisation revealed?

Answer 13

IgG crystallisation

Ig fold:
Two sheets of antiparallel B-strands, connected by unstructured loops - identified as CDRs which form part of the antigen-binding site.

Corresponds to hypervariable regions in the primary sequence

Question 14

Humanized antibodies

Answer 14

CAMPATH - anti-CD52 antibodies raised in mice, but with CDRs grafted onto human IgGs

Transfers antigen specificity but reduces immunogenicity

Question 15

Phage display

Answer 15

1. Modify phage DNA to display one candidate peptide as a fusion protein with the viral coat protein, for all peptides within a large library
2. Expose phage library to immobilised target protein, and wash off any non-specifically or loosely bound phage (select for tight binding)

Phages that bind strongly are eluted, the DNA is sequenced

Revise peptide library, or further improve affinity using dirty PCR

Question 16

Phage display for antibodies

Answer 16

Can use scFv for phage display, and randomising CDRs

HUMIRA - anti-TNFa

Question 17

Ribosome display

Answer 17

in vitro system - DNA library transcribed in vitro, mRNA translated by ribosomes.

mRNA-ribosome-protein complex undergo selection by binding an immobilised target

Tight-binders are eluted, mRNA isolated and reverse transcribed to DNA

in vitro, removes unwanted selection pressures (eg. expression, degradation, transformation efficiency)

Question 18

Alternative scaffolds

Answer 18

Similarly with a structured core and unstructured loop - eg. fibronectin, DARPins
Affimers based on cystatin scaffold - small size, thermal stability

Question 19

Directed evolution by screening

Answer 19

Error-prone PCR, then screen cell lysate (or purified protein) under desired conditions

Much lower throughput than phage display

Nitrobenzyl esterase - engineered to have much higher activity and thermal stability
New salt bridges, H-bonds, tighter packing of secondary structure. Many mutations not in the active site

Question 20

Protein engineering

Answer 20

T4 lysozyme

Mutations to Cys - introduce new S-S bridges in appropriately-positioned residues.

Add negative residues to N-terminal of a-helices to neutralise dipole.

Improves thermal stability. But S-S bridges resulted in decreased stability in reducing conditions

Question 21

Levinthal’s paradox

Answer 21

Many possibilities of protein folding - but correct folding occurs almost instantly.

Molten globules - fast-forming folding intermediates, usually secondary structures, that limit the number of folding possibilities

Question 22

Chaperones

Answer 22

DnaJ/DnaK - DnaJ binds hydrophobic residues, prevents clustering until complete folding unit is synthesised. Dissociation is caused by ATP hydrolysis by DnaK

GroEL/GroES. GroEL forms two heptameric rings, lined with hydrophobic residues. GroES caps the cavity, recruits ATP. Conformational change causes cavity to become hydrophilic, and folding of the sequestered protein occurs. ATP hydrolysis enables opening of the cavity and release of folded protein