chymotrypsin (serine protease)

Question 1

Enteropeptidase

Answer 1

starts the activation
by converting trypsinogen to trypsin

Question 2

Five major pancreatic proteases

Answer 2

Trypsin
Chymotrypsin
Elastase
Carboxypeptidase A
Carboxypeptidase B

Question 3

Endopeptidases

Answer 3

Trypsin
Chymotrypsin
Elastase

Question 4

Exopeptidases

Answer 4

Carboxypeptidase A
Carboxypeptidase B

Question 5

Specificity of chymotrypsin: peptidase cleavage sites

Answer 5

Trp
Tyr
Phe
Met
Leu

Question 6

Key active site residues

Answer 6

His 57, Asp 102 and Ser 195– catalytic triad

SERINE plays such an
important role in the active site,
CHYMOTRYPSIN is a SERINE PROTEASE

Question 7

Ser 195

Answer 7

provides the nucleophile
(O atom)

Question 8

His 57

Answer 8

acts as a base to activate Ser

Question 9

Asp 102

Answer 9

stabilises His 57

Question 10

step 1

Answer 10

His 57 activates Ser 195, extracts a
proton from Ser

Oxygen on Ser now forms a new
COVALENT bond with the carbonyl
carbon in the polypeptide

Question 11

step 2

Answer 11

ser and his generate a strong nucleophilic alkoxide ion on ser which attacks peptide carbonyl forming
unstable terahedral acyl-enzyme

shortlived negative charge on o that is stabilised by H bonding

Question 12

step 3

Answer 12

re-from carbonyl, PEPTIDE BOND breaks, release small peptide in which His 57 now donates the proton it gained to the N in the peptide bond

But…the rest of the peptide is still
attached covalently to the enzyme!
(Acyl-enzyme intermediate)

Question 13

step 4

Answer 13

H2O comes in and is
deprotonated by His 57 and
generates a strong nucleophile

Attacks the ester linkage of the
acyl-enzyme- second tetrahedral

Question 14

step 5

Answer 14

short lived intermediate, forms second product - carboxykate anion and displaces ser

his donates proton back to serine, restore activity, second half of peptide released

Question 15

Protein/enzyme engineering

Answer 15

creating new proteins through manipulation of the DNA encoding the
protein

Question 16

create a single protein/enzyme with
enhanced or altered specificity or function

Answer 16

Proteases

Question 17

design entire metabolic pathways to
create new products

Answer 17

Stevia

Question 18

Strategies for protein engineering

Answer 18

Directed evolution
rational design
semi-rational design

Question 19

Directed evolution

Answer 19

moves the evolution
process into the laboratory and
speeds it up.
intended variation of protein
sequences at a defined level of
randomness

Question 20

directed evolution advantages

Answer 20

No prior knowledge of
protein/enzyme structure is
required
* Mutate entire protein  could find
distant regulatory sites

Question 21

directed evolution disadvantages

Answer 21

Large library size required
* High-throughput screening can be
difficult, particularly for enzymes

Question 22

Directed evolution of enzymes

Answer 22

1) Identification of a suitable starting
enzyme for the chosen task
2) DNA-sequence library construction
to cover a well-chosen subsets of
sequence space
3) Identification of selected criteria
that will lead to enhanced or new
functions AND methods for
selection of optimized enzyme
variants
4) Set up selection criteria with
increased stringency
5) Repeat as many rounds as need to
reach target level of enzyme
performance

Question 23

What is phage display?

Answer 23

A method to construct large peptide
libraries, most commonly using M13
filamentous bacteriophage (phage)

Question 24

viral coat of M13 consists of
five different capsid proteins- name?

Answer 24

pVIII (2700 copies)
* Minor capsid proteins of:
* pIII and pVI at one end of the
phage;
* pVII and pIX at the other end

Question 25

MABS

Answer 25

For phage display, Fab
(fragment antigen
binding) or scFv (single
chain fragment variable)
parts of the antibody are
fused to pIII in M13
phage

Question 26

nanobodies

Answer 26

VHH single domain antibody

Question 27

nanobody purpose in vitro

Answer 27

design antibodies to ‘trap’ membrane proteins
in specific conformations
 Developed an in vitro selection for synthetic
nanobodies: sybodies