Lysozmes and Serine Proteases

Question 1

What is a lysozyme?

Answer 1

An enzyme that destroys bacterial cell walls

Working at a pH range of 3-8

Question 2

How does a lysozyme work?

Answer 2

In bacteria
It hydrolyses the b(1-4) glycosidic linkages of N-acetylmuramic acid to N-acetylglucosamine in the cell wall of peptidoglycans

In fungi
It hydrolyses the b(1-4) linked poly N-acetlyglucosamine in the cell wall of chitin

Question 3

Give an example of a lysozyme?

Answer 3

Hen egg white lysozyme

D-ring with half-chair formation
Small protein - 14.3 kDa
Single polypeptide chain of 129 amino acid residues
Internally crosslinked by 4 disulfide bridges
increases the rate of reaction 10^8 fold greater
Active site is a prominant cleft - that traverses one face of the molecule

Question 4

What is the reaction that a lysozyme catalyses?

Answer 4

Hydrolysis of an acetal to a hemiacetal

1. protonation of an acetals oxygen
2. cleavage of its C-O bond forming an alcohol and a resonance stablised carbocation (oxonium ion)
3. Adding water to the oxonium ion forms the hemiacetal and regenerates the H+ catalyst

Question 5

What are lysozymes catalytic residues?

Answer 5

Glu35 and Asp52

Glu35 is in a relatively nonpolar pocket, remaining protonated
Therefore it can act as an acid catalyst

Asp52 is surrounded by polar residues forming a complex hydrogen-bonded network
Therefore Asp52 can work electrostatically to stabilise the oxonium ion

Question 6

What is the lysozymes catalytic mechanism?

Answer 6

1. enzyme attaches to a hexose in the bacterial cell wall, moving the D residue towards the half-chair conformation
2. General acid-catalysis, Glu35 tranfers a proton, facilitating cleavage = oxonium ion transition state, this is stabilised by Asp52
3. Covalent catalysis, Asp52 nucleophilically attacks C1 on the D ring to form a glycosyl-enzyme intermediate
4. Water replaces the E-ring product in the active site
5. Base-catalysis, Glu35 helps the hydrolysis of the covalent bond, regenerating the active site groups and releasing the D-ring product

Question 7

What is an inhibitor to a lysozyme?

Answer 7

The δ-lactone a transition state analog of (NAG)4
As the compounds lactone ring has the half chair formation (geometrically similar to the oxonium ion) it can bind tightly to the lysozyme

Question 8

How was evidence for covalent catalysis discovered for the lysozyme?

Answer 8

1. Substitute F at C2 (remove electron withdrawing effects)
2. Mutate Glu35 to Gln (removing acid-base catalysis)
3. Substitute another F at C1

These changes increase the rate of formation of the covalent intermediate

Question 9

What are serine proteases?

Answer 9

They cleave proteins by aid in hydrolysis of peptide bonds, and their reaction involves a very reactive serine residue
They do not cleave at the serine site

Synthesised in the pancreas
Secreted into the duodenum

Question 10

What are serine proteases involved in?

Answer 10

Development - they can prevent webbed hands (body morphology)
Inflammation pathways
Blood clotting

Question 11

What are the serine proteases we need to know?

Answer 11

Chymotrypsin
Trypsin
Elastase

Question 12

Where does each serine protease cleave a protein?

Answer 12

Chymotrypsin - cleaves next to a bulky hydrophobic residue

Trypsin - cleaves next to a positive residues

Elastase - cleaves next to a small neutral residue

Question 13

How was the reactive serine residue on chymotrypsin discovered?

Answer 13

Reaction with diisopropylphosphofluoridate (DIPF) irreversibly inactivates the enzyme
The other serine residues don’t react with DIPF) therefore it must by the Ser195 in the active site

Question 14

What else can DIPF be used for?

Answer 14

Used as an enzyme-inactivating agent as a potent nerve poison
It can inactivate the neurotransmitter acetylcholinesterase

Question 15

Name some other neurotoxins?

Answer 15

Parathion and malathion - insecticides

Sarin - nerve agent

Question 16

How was the reactive histidine residue discovered in chymotrypsin?

Answer 16

Affinity labelling

TPCK a substrate analog with a reactive group binds to the enzyme and only reacts with His57 - inactivating the enzyme

Question 17

What is the general structure found of the serine proteases?

Answer 17

Two domains
Extensive antiparallel beta sheets in a barrel-like arrangement but few helices
40% similar - but have different specificities
Catalytic triad

Question 18

What is the Catalytic triad?

Answer 18

3 residues forming a hydrogen-bonded constellation within a solvent inaccessible pocket

Ser195
His57
Asp102

Question 19

What is contained within the specificity pocket of each serine protease?

Answer 19

Chymotrypsin - Gly 226 on the left, Gly 216 on the right and Ser 189 at the bottom

Trypsin - Gly 226 on the left, Gly 216 on the right and Asp 189 at the bottom

Elastase - Val 226 on the left, Thr 216 on the right and nothing at the bottom

Question 20

What do the serine protease exhibit?

Answer 20

Divergent and convergent evolution

Question 21

What is divergent and convergent evolution?

Answer 21

Divergent evolution - a duplication of an ancestral serine protease - that would likely be very specific

Convergent evolution - where for example subtilisin or carboxypeptidase II come together to form a new enzyme over many years
The residues don’t have the same order/sequence but when they come together the catalytic triad will meet

Question 22

What is the catalytic mechanism of a serin protease e.g. chymotrypsin?

Answer 22

1. Ser 195 nucleophilically attacks the scissile peptide’s carbonyl group using covalent catalysis to form a tetrahedral intermediate
   This also uses base catalysis from His57 imidazole ring
2. General acid catalysis helps the breakdown of the tetrahedral intermediate to the acyl-enzyme intermediate
3. The amine group is released and replaced by water
4. General base catalysis and nucleophilic attack to form a 2nd tetrahedral intermediate
5. General acid catalysis helps the breakdown of the intermediate to the carboxyl product and the active enzyme

Question 23

How do serine proteases preferentially bind to the transition state?

Answer 23

The rearrangement of the tetrahedral intermediate from sp2 hybridized to sp3 hybridized causes the oxygen on the COO of the scissile peptide to move deeper into the active site - not occupying the oxyanion hole
It now forms two hydrogen bonds with the enzyme
The tetrahedral distortion allows the formation of an unsatisfied hydrogen bond between the enzyme and a NH group of the substrate

Question 24

What can also stabilise the transition state?

Answer 24

Low-barrier hydrogen bonds (LBHBs)
These are additional hydrogen bonds not through the oxyanion hole
When pKs of hydrogen bonding donor/acceptor are nearly equal the atom becomes almost equally shared

Question 25

Describe low-barrier hydrogen bonds?

Answer 25

Short and strong
High free energy of formation (measured in the gas phase)
They can exist in non-aqueous sites of enzymes (water molecules have good acceptors/donors)

Question 26

Why are inhibitors used? Example?

Answer 26

Inhibitors can be used to prevent proteins from digesting the tissues they are in

BPTI - bovine pancreatic trypsin inhibitor

Question 27

How does BPTI work?

Answer 27

It inhibits prematurely activated trypsin in the pancreas from digesting the organ

A Lys side chain of BPTI occupies the trypsin specificity pocket (not a substrate)
A proteolytic reaction doesn’t take place due to the rigidity of the complex and is so tightly sealed that the leaving group can’t leave and water can’t enter

Question 28

What are zymogens?

Answer 28

Inactive enzyme precursors

Meaning proteolytic enzymes are usually biosynthesised from larger inactive precursors (proenzymes)

Question 29

What would happen if we didn’t have zymogens?

Answer 29

If the enzymes were synthesised in their active form, they would digest the tissues that synthesised them

Acute pancreatits - the premature activation of the digestive enzymes (usually by pancreatic trauma)

Question 30

What is the zymogen of trypsin?

Answer 30

Trypsinogen
It is activated when it enters the duodenum (from the pancreas)
Enteropeptidase cleaves Lys 15 - Ile 16 peptide bond to form trypsin

The presence of trypsin also catalyses trypsinogen activation - generating more trypsin = autocatalytic

Question 31

What are the zymogens of other hydrolytic enzymes?

Answer 31

Chymotrypsinogen -> chymotrypsin
Proelastase -> elastase
Procarboxypeptidase A/B -> carboxypeptidase A/B
Prophospholipase -> phospholipase

Question 32

What activates each hydrolytic enzyme from their zymogen?

Answer 32

Trypsin activates them all

Therefore it needs to be carefully regulated and it is essential it is not activated in the pancreas

Question 33

Why is sequential proenzyme activation useful?

Answer 33

Generating large quantities of active enzymes e.g.

When a clot forms from a damaged blood vessel, fibrin network traps additional blood cells
Thrombin activates fibrin from fibrinogen
Many factors are activated to produce fibrin known as a coagulation cascade

Question 34

Why are zymogens inactive?

Answer 34

They have distored active sites
The specificity pocket and oxyanion hole are improperly formed therefore no ES complex or stablised tetrahedral intermediate