Exam Essay prep Flashcards
First paragraph: Define Covalent catalysis
Cat
Translated
internship
Substitution
Covalent catalysis is one of the four strategies that an enzyme uses to catalysis a specific reaction.
This process involves a substrate or a part substrate which is covalently bound to an enzyme, this is a transient interaction forming a reactive intermediate
Second paragraph: Explain the reaction for covalent bond
Nuclear Electrocuted siked loving nukes unstable
In this reaction, the enzyme contains a reactive group, either a nucleophilic or electrophilic residue.
This reacts with the substrate by a nucleophilic or electrophilic attack.
second paragraph part 2: Explain Nucleophilic attack
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Loving nukes
unstable
Nucleophilic attack involves the attack of a positively charged (or partially positively charged) atom or group of nucleophiles
Third paragraph: Explain what the formation of the covalent bond provides
Configuration
increases
flavour
Because of the formation of the covalent bond, it provides chemistry that enhances reaction rate making the reaction more favourable.
Third paragraph part 2: Explain the subsequent steps in the catalytic mechanism transfer reactive intermediate to a second substrate
Form Reaction Favourable Next Cat A Equals X A X B
Subsequent steps in the catalytic mechanism transfer reactive intermediate to a second substrate.
The group transfer from substrate Ax involves the interaction of the enzyme with the initial substrate to form a substitution enzyme intermediate, enzyme X.
Enzyme X will the interact with substrate B leading to another group transfer to substrate A, forming the final product Bx, releasing the enzyme for catalysis. This process can be seen in the equation below.
Enzyme + Ax -> Enzyme- X + A -> Enzyme – X + B -> Enzyme + Bx
Fourth paragraph: How are intermediates involved
Free Energy State form route low state speed Cove active diffuse speed invest second reaction
You need an input of free energy to achieve transition state, however the formation of the intermediate gives a chemical route allowing for the lower of transition state, speeding up the reaction.
Covalent intermediates are held in the active sites which stops it from diffusing away, meaning it doesn’t need to be defused back in, therefore it also speeds up the reaction because the energy doesn’t need to be used to diffuse it again but can be invested in the second transition of the reaction.
Fifth paragraph: What are Serine proteases?
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A good example of this type of mechanism is Serine protease, this is part of a characterised family of enzymes meaning they are a group of enzymes that are all sequentially homologous and closely similar, with an overall three-dimensional geometry.
This family includes trypsin, chymotrypsin (both found in the digestive tract), Elastase (involves in the defence mechanism for white blood cells), thrombin, subtilisin, plasmin, TPA. All these characterized family of enzymes all exploit the process of covalent catalysis seen in protease.
sixth paragraph: Explain the architecture of serine protease
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Because Serine proteases are homologous this means they all share common architecture which consists of a common 3D structure which is made up of a beta-barrel domain, converging on a catalytic active site.
Serine proteases have an active site residue located in a cleft on the surface of the enzyme, the cleft has loops forming walls of the cleft, this is designed to help substrate binding. Multiple sub-sites are formed on the cleft to help with substrate binding.
The cleft is formed between 2 beta barrels, this structure is known as the chymotrypsin fold. They all share three crucial residues (His, Asp and Ser), these residues are involved in catalytic mechanism
Seventh paragraph: Explain the first 3 steps of the reaction mechanism Proximity Nuketown State stable form cove
1)
Interaction, recognition, coordination,pep, bond,cleavage
2) Hi bond sir his sir nuke town pepsi bond
3)
tetra, cleavage, Pepsi, bond, unstable,interact, ox,hole, introduction, ace, positive, his
The reaction mechanism proposed for serine protease includes the proximity, nucleophilic attack, transition state stabilisation and the formation of a reactive covalent intermediate.
The first step in the reaction mechanism is the interaction with substrate recognition site, coordinates peptide bond for cleavage. Followed by a hydrogen bonding between Ser 195-OH and His57 resulting in activation of Ser-195 for nucleophilic attack of a peptide C=O bond. The third step then begins with the formation of first tetrahedral intermediate, involving the cleavage of the peptide bond but because this reaction is unstable, this is stabilised by an interaction with oxyanion hole as well as the introduction of Asp102 to stabilise the positive charge on the His57.
Eighth paragraph: Explain 4th, 5th, 6th and 7th steps of the reaction mechanisms
4) Pep,fragile,lost, cove, axel,High, Esther, bond ;hydro lis, pepsi, bond.
5) Promote, water, bond, his, increase, nuclear felicity, O, Nuketown, Esther, carb neil
6) form, tetra, unstable, stable, oxy, hole, ace, positive, his, Carb, ox, diffuse, round, cat
7) Carb, ox, diffuse, round, cat
The fourth step in the proposed reaction mechanism for serine proteases includes the first peptide fragment to be lost, forming a covalent acyl enzyme intermediate to be formed. The intermediate formed now has a highly active ester bond and is more readily hydrolysed than peptide bond.
The fifth step in the mechanism is water being promoted by H-bonding to His57, this reaction cases an increase in nucleophilicity of O atom and a nucleophilic attack on ester carbonyl carbon atom.
The sixth step involves the formation of second tetrahedral intermediate, the result is unstable meaning it is then stabilised by interaction with oxyanion hole formed previously formed as well as the Asp102 which is around to stabilise the positive charge on His57. The final step in the reaction mechanism is the carboxyl component being diffused away, leaving an enzyme that is ready for another round of catalysis.
Ninth paragraph: Explain Burst Kinetics and its evidence
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There are many different experimental evidences supporting the proposed reaction mechanism.
This includes Burst Kinetics which is another form of enzyme kinetics. Burst kinetics is the large initial velocity exhibited during the process of adding an enzyme to a substrate. The large initial velocity is then levelled off once all the enzymes have been saturated leading to enzyme velocity linearly increasing.
The idea of Burst kinetics was introduced over 50 years ago, it showed that when an enzyme has two products, it may show a rapid production of one of the products in the pre-steady-state regime. Later work showed that it is quite common for a hydrolytic enzyme with a ordered ping-pong-bi-bi reaction sequence to have this.
Tenth paragraph: Explain Modification of Serine 195
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Modification of Serine 195 is another example. An experiment involving Chymotrypsin a serine protease enzyme that cleaves the peptide bonds selectively on the carboxy terminal side of the larger hydrophobic amino acids such a tryptopham and tyrosine etc. Chymotrypsin employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate, the nucleophile becomes covalently attached to the substrate briefly in the course of catalysis.
The experiment comes with the knowledge that chymotrypsin contains an extraordinarily reactive serine residue, this is then treated with organoflurophosphates such as diisopropylphosphoflouridate, this inactivates the enzyme irreversibly. With the Serine 195 modified resulting in a loss of enzyme activity, this suggests that this unusually reactive serine residue plays a central role in the catalytic mechanism of chymotrypsin.
11th paragraph: Explain the modification of Histidine 57
change History chemotherapy Change Certain Imu
Modification of Histidine 57 is another example.
Histidine 57 is essential for activity in the chymotrypsin, the idea discovered through kinetic studies and chemical modification. Specific methylation of imidazole devised by Naka-gawa and Bender shows that a clean and minor modification of the active site is enough to inhibit the enzyme entire, proving this.
11th paragraph (part 2): Explain site-directed mutagenesis of Asp102
Genesis Ace Craik Rutter Genesis Mutant Trip Margarine Mutant possessed Hydro native Cat immobile his hydro bond cat triad
. Site-directed mutagenesis of Asp102 is another example, in 1987 Charles Craik, William Rutter and their colleagues used site-directed mutagenesis to prepare mutant trypsin with asparagine in place of Asp-102.
Mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp102 is essential for catalysis and that its ability to immobilize and orient His57 by the formation of a hydrogen bond is crucial to the function of the catalytic triad.
