Proteases Flashcards
What are the five different proteases?
Serine proteases- covalent attack by a serine hydroxyl,
Aspartic proteases- attack by polarised water,
Cysteine protease- covalent attack by a cysteine thiolate,
Threonine protease- covalent attack by a Threonine hydroxyl,
Metallo protease- attack by polarised water.
List some processes that proteases are involved in.
Ovulation, Blood clotting, Fertilisation, Embryogenesis, Development, Apoptosis, Cell cycle, Immunity, Complement activation
What is degradation associated with?
What is limited proteolysis associated with?
Degradation is associated with loss of function,
Limited proteolysis is associated with gain of function.
Digestion of the below polypeptide with chymotrypsin results in what?
O
R - C - N - R
H
O H+
R - C - O^- H - N - R
H
Polypeptide-acid. Amine-polypeptide
Serine proteases possess what four key features that allow them to accelerate peptide bond hydrolysis?
They possess a catalytic triad (Asp, His, Ser),
They possess an oxyanion hole (Main chains possess Gly and Ser),
They possess a substrate specificity pocket,
They possess a substrate main chain binding site-non-specific.
What are the similarities and differences between chymotrypsin like serine proteases?
They all possess a catalytic triad,
The residues involved in active site formation are conserved,
There are large variations in surface expressed loop regions.
How are serine proteases activated?
Why is this strategy used?
They are activated by limited proteolysis. Serine proteases are synthesised as inactive zymogens, with limited proteolysis converting them into the active mature enzyme.
This is used to prevent premature activity.
In metallo proteases how is activity prevented in zymogens?
In serine proteases how is activity prevent in zymogens?
What is the name of the segment cleaved in zymogens, what can it aid in?
What terminal is the segment to be cleaved?
In metallo proteases there is physical blocking of the active site,
In serine proteases the active site is deformed.
The pro-peptide or prosegment. It can aid in stability of the protein it inhibits.
The pro-peptide is N-terminal on the protein.
What enzyme activates chymotrypsinogen?
How does this enzyme activate chymotrypsinogen?
Trypsin activates chymotrypsinogen,
It activates it through cleaving between arg15 and ile16. The NH3^+ of the ile16 residue then forms a salt bridge with asp194 in the interior of the protein.
Zymogen activation of chymotrypsinogen leads to what conformational changes?
The salt bridge forming between ile16 and asp194 causes the backbone of the protein to rotate. This puts the NH groups of gly193 and ser195 to be in the correct orientation to form the oxyanion hole. Met192 then shifts to open the substrate specificity pocket. After this all the catalytic features are in place and the enzyme is active.
What key features of the active enzyme are present in the zymogen?
In the zymogen there is a catalytic triad but no oxyanion hole. Also there is a substrate specificity pocket but it is incorrectly formed. Overall 2 of the 4 features are present in the zymogen.
Describe where chymotrypsin is located and what it catalyses.
Chymotrypsin is located in the intestine. It is an endopeptidase that hydrolyses bonds adjacent to the carboxyl group of aromatic amino acids such as phenylalanine, tyrosine and tryptophan. Chymotrypsin can also catalyse the hydrolysis of artificial substrates.
The hydrolysis of what substrate provided insight into the mechanism of peptide and ester hydrolysis?
P-nitophenylacetate.
How does enzyme catalysed hydrolysis work?
What does these specific phases of reaction suggest?
It occurs in two distinct phases. There is an initial rapid burst if phenolate production which is followed by a slower steady state rate of phenolate production.
These two phases suggest that the reaction proceeds via a reaction intermediate.
How can the burst phase and steady state phase be explained, what with classic michaelis menten kinetics suggesting that the reaction should be steady state?
The behaviour of chymotrypsin can be explained through a intermediate forming. For normal proteases E + S –> ES –> E +P1+P2
However for chymotrypsin the reaction is:
E + S –> ES –> E-P2 + P1 –> E + P2 + P1
The intermediate being the enzyme bound to P2 while P1 dissociates.
Describe the burst phase and steady state phase in relation to chymotrypsin and P-nitrophenylacetate.
Firstly E-acetate is formed rapidly, this being the burst phase. The burst is caused by the high amount of P-nitrophenylate that is produced, which is equal to the amount of active enzyme. This is the acylaction step. After this the acetate dissociates from the enzyme and the result is steady state reaction kinetics from then onwards. The release of acetate is the rate defining step of the reaction, this steady state being the deacylation step.
What property of ser195 is increased by what?
The main chain amides of what residues form the oxyanion hole which does what?
The nucelophilicity of ser195 is increased via a Hydrogen bond to His57. His57 is polarised by asp102 so that it can become an efficient H^+ shuttle.
The main chain amides of gly193 and ser195 form the oxyanion hole which stabilises the build up of charge in the tetrahedral intermediate.
How was the acyl-enzyme intermediate E-P2 identified?
What residue was found to be covalently attached to the acyl group.
What is the rate limiting step for ester hydrolysis and why?
What is the rate limiting step for amide hydrolysis and why?
It was found to be stable at a pH of 3, after which it was isolated and characterised by crystallography.
For ester hydrolysis the deacylation step is the rate limiting step, this is due to esters being good leaving groups after the reaction in chymotrypsin. For amide hydrolysis the acylation step is the rate limiting step, this is due to amides being poor leaving groups after the reaction in chymotrypsin.
