Lysozmes and Serine Proteases Flashcards
What is a lysozyme?
An enzyme that destroys bacterial cell walls
Working at a pH range of 3-8
How does a lysozyme work?
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
Give an example of a lysozyme?
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
What is the reaction that a lysozyme catalyses?
Hydrolysis of an acetal to a hemiacetal
- protonation of an acetals oxygen
- cleavage of its C-O bond forming an alcohol and a resonance stablised carbocation (oxonium ion)
- Adding water to the oxonium ion forms the hemiacetal and regenerates the H+ catalyst
What are lysozymes catalytic residues?
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
What is the lysozymes catalytic mechanism?
- enzyme attaches to a hexose in the bacterial cell wall, moving the D residue towards the half-chair conformation
- General acid-catalysis, Glu35 tranfers a proton, facilitating cleavage = oxonium ion transition state, this is stabilised by Asp52
- Covalent catalysis, Asp52 nucleophilically attacks C1 on the D ring to form a glycosyl-enzyme intermediate
- Water replaces the E-ring product in the active site
- Base-catalysis, Glu35 helps the hydrolysis of the covalent bond, regenerating the active site groups and releasing the D-ring product
What is an inhibitor to a lysozyme?
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
How was evidence for covalent catalysis discovered for the lysozyme?
- Substitute F at C2 (remove electron withdrawing effects)
- Mutate Glu35 to Gln (removing acid-base catalysis)
- Substitute another F at C1
These changes increase the rate of formation of the covalent intermediate
What are serine proteases?
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
What are serine proteases involved in?
Development - they can prevent webbed hands (body morphology)
Inflammation pathways
Blood clotting
What are the serine proteases we need to know?
Chymotrypsin
Trypsin
Elastase
Where does each serine protease cleave a protein?
Chymotrypsin - cleaves next to a bulky hydrophobic residue
Trypsin - cleaves next to a positive residues
Elastase - cleaves next to a small neutral residue
How was the reactive serine residue on chymotrypsin discovered?
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
What else can DIPF be used for?
Used as an enzyme-inactivating agent as a potent nerve poison
It can inactivate the neurotransmitter acetylcholinesterase
Name some other neurotoxins?
Parathion and malathion - insecticides
Sarin - nerve agent
How was the reactive histidine residue discovered in chymotrypsin?
Affinity labelling
TPCK a substrate analog with a reactive group binds to the enzyme and only reacts with His57 - inactivating the enzyme
What is the general structure found of the serine proteases?
Two domains
Extensive antiparallel beta sheets in a barrel-like arrangement but few helices
40% similar - but have different specificities
Catalytic triad
What is the Catalytic triad?
3 residues forming a hydrogen-bonded constellation within a solvent inaccessible pocket
Ser195
His57
Asp102
What is contained within the specificity pocket of each serine protease?
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
What do the serine protease exhibit?
Divergent and convergent evolution
What is divergent and convergent evolution?
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
What is the catalytic mechanism of a serin protease e.g. chymotrypsin?
- 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 - General acid catalysis helps the breakdown of the tetrahedral intermediate to the acyl-enzyme intermediate
- The amine group is released and replaced by water
- General base catalysis and nucleophilic attack to form a 2nd tetrahedral intermediate
- General acid catalysis helps the breakdown of the intermediate to the carboxyl product and the active enzyme
How do serine proteases preferentially bind to the transition state?
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
What can also stabilise the transition state?
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
Describe low-barrier hydrogen bonds?
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)
Why are inhibitors used? Example?
Inhibitors can be used to prevent proteins from digesting the tissues they are in
BPTI - bovine pancreatic trypsin inhibitor
How does BPTI work?
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
What are zymogens?
Inactive enzyme precursors
Meaning proteolytic enzymes are usually biosynthesised from larger inactive precursors (proenzymes)
What would happen if we didn’t have zymogens?
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)
What is the zymogen of trypsin?
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
What are the zymogens of other hydrolytic enzymes?
Chymotrypsinogen -> chymotrypsin
Proelastase -> elastase
Procarboxypeptidase A/B -> carboxypeptidase A/B
Prophospholipase -> phospholipase
What activates each hydrolytic enzyme from their zymogen?
Trypsin activates them all
Therefore it needs to be carefully regulated and it is essential it is not activated in the pancreas
Why is sequential proenzyme activation useful?
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
Why are zymogens inactive?
They have distored active sites
The specificity pocket and oxyanion hole are improperly formed therefore no ES complex or stablised tetrahedral intermediate
