Enzymes and their Mechanisms Flashcards
Acid and base definition
Acid - Proton donor
Base - Proton acceptor
Rearranged Henderson-Hasselbach equation to compare ratio of [HA] to [A-]
pH - pKa= log10([A-]/[HA])
Factors that affect acidity of a compound (Y-H)? (5 factors)
Strength of Y-H bond
Electronegativity of Y
Factors that stabilise Y- vs Y-H
Nature of solvent
Temperature
Lower pKa vs Higher pKa in terms of acid and base strength?
Lower pKa means a stronger acid and weaker base
Higher pKa means inverse
Rank acidity of carboxyl, hydroxyl and methane and why?
Lowest acidity
- Methane
- Hydroxyl
- Carboxyl
Highest acidity
Greater polarisation in the molecules which are stronger acid - Lose H more easily
Tautomer vs Resonance structure?
Tautomer - Atoms move within structure
Resonance structure - Atoms don’t move, only electrons move
What is acid-base catalysis?
When a proton is transferred to or from the transition state
What does the endonuclease RNAse A do and how specific is it?
Cleaves ssRNA into smaller fragments
Very specific as it cuts after a pyrimidine base; Must be a recognition site 9
What are the 2 steps of the RNAse reaction?
Formation of 2’-3’ cyclic intermediate
Cleavage of cyclic intermediate into final compound
What does the pH profile of the enzyme Vmax suggest about the RNase active site structure? - Give characteristics
2 Histidine residues
- One acts as an acid (protonated)
- One acts as a base (deprotonated)
What does modification of RNAse tell us about the active site histidines?
They are close together in the active site - Only one or the other is modified
One acts as an acid, one acts as a base
Structure of RNAse - 3 key points
Structure consists of:
- Anti Parallel β-sheet - 3-Stranded and V-Shaped
- 3 short α-helices
Peptide chain cross-linked by 4 S-S bridges
Active site is a deep cleft containing essential residues
What are all 4 residues in the active site of RNAse? (4th is more general)
- (dont confuse with specificity pocket residues)
His12 and His 112 are the catalytic duo
Lys41 stabilises -vely charged phosphate in intermediate
Basic residue for RNA binding
What are the 3 residues in the specificity pockets of RNAse?
How is purine binding prevented?
Phe120 has Van Der Waal contact with RNA base
Ser123 and Thr45 forms H-bonds
Pockets too small for purines
Look at RNAse A Reaction Mechanism - Describe mechanism in words
His12 acts as base and His119 acts as an acid to convert nucleic acid to cyclic intermediate
Histidines roles are reversed to hydrolyse cyclic intermediate
At the end of the cycle, His residues returned to initial configuration
What is the scissile bond?
Bond at which the hydrolysis cleavage occurs
Why do proteases have multiple specificity pockets?
So they can select for certain types of side chains
What are the pockets of the following enzymes specific too?
- Chymotrypsin
- Elastase
- Trypsin
Chymotrypsin - Big and hydrophobic molecules
Elastase - Small and hydrophobic
Trypsin - Basic/long +ve
How can proteases be extra specific?
Proteases can have elaborate side chain and main chain recognition sites, limiting the number of substrates that can bind
How are proteases regulated to prevent excess protein hydrolysis and damage?
Produced as inactive enzyme - Zymogen
Key facts about chymotrypsin (3 facts) (hint - serine protease)
Produced as precursor called Chymotrypsinogen
Cleaves large aromatic residues
Essential Ser195 (serine protease) and His57 residues
How is Chymotrypsinogen converted to Chymotrypsin?
Through proteolytic cleavage in an activation cascade; Non-reversible
Cleaves long polypeptide into multiple connected by S-S (tertiary)
Structure of Chymotrypsin (hint - Types of structures and residues)
- What key thing is formed by the residues?
2 β-barrels, each formed from 6 anti-parallel beta-strands
In deep cleft - Essential His57 and Ser195 residues with Asp 102; Form CATALYTIC TRIAD
How do the Asp, His and Ser interact with eachother to ensure catalytic activity in Chymotrypsin?
How is Asp less important than the other 2?
-ve Asp stabilises formation of +ve His, allowing it to grab proton from Ser, making it nucleophilic and high reactive
Reaction can still occur without Asp, however catalytic rate is massively reduced
What is the oxyanion hole?
What is its purpose in the reaction?
Region in the active site where the backbone amide hydrogens of Ser195 and Gly193 point into the active site
The hydrogens stabilise oxyanion on the intermediate and enable a specific electron flow - Increases activity massively
Look at Chymotrypsin mechanism
How do different serine proteases change their specificity? (2 ways)
Altering pocket size through having residues sticking out into the pocket
Different residues at bottom of pocket to alter what can bind
How is Chymotrypsinogen activated upon cleavage by Trypsin? (hint - think about what is needed structurally for the enzyme to work)
New terminal NH3 pairs to Asp 194 altering the conformation of the main chain residues between 193 and 195
This creates the correct geometry to form the catalytic triad and the oxyanion hole
What is a proteasome and what type of peptidase does it contain?
Complex containing multiple protein subunits with active sites mechanistically belonging to the Threonine Hydrolase family
As the proteasome can cause cancer, how can boron containing compounds inhibit this? (hint - talk about mechanism and boron geometry using necessary jargon)
Boron containing compounds exhibit apoptosis in cells, helping treat cancer
Boron is electron deficient and forms a covalent bond with active site nucleophiles; Go from trigonal Sp2 to tetrahedral Sp3
This creates a -vely charged boron atom bound to the enzyme, inhibiting its activity
How is cysteine different to serine and threonine?
How is it a better acid?
Cysteine has an SH group whereas the others have an OH group
Cysteine has a lower pKa than the others; Can lose its proton more easily
- Due to S being less electronegative, so it holds onto the H less
Look at Cysteine protease mechanism
How is the cysteine protease mechanism similar to deamidation?
What organism often utilises this?
Both utilise a His/Cys dyad to enable hydrolysis of C-N adjacent to carbonyl
Bacteria use deamidation enzymes as toxins
How is BPSL1549 protein of Burkholderia pathogen similar to CNF1? (hint - spatial conservation)
They don’t share sequence similarity but they do share structural similarity
- Both share the same spatial conservation of His/Cys pair
What does CNF1 do?
Deamidates a key glutamine in the family of GTPases
This blocks GTP hydrolysis, and therefore GAP-dependent deactivation of the GTPases
Results in remodelling of actin cytoskeleton
What is the key characteristic of the Enolase superfamily? (hint - divalent)
What is its role?
Utilise Mg2+ ion to facilitate catalysis
Stabilises the intermediate with its +ve charge - Coordinates with oxygens atoms
What are the 2 key domains in Enolase enzymes and what are their functions?
Where does substrate binding occur?
Barrel domain - Residues involved in acid/base catalysis found here
Capping domain - Determines specificity
Substrate bind at interface of capping and barrel domains
What is an enolate anion?
Carbonyl with adjacent C with acidic H removed; Negative charge can move between C and O
Look at Mandelate Racemase mechanism?
What is the end result?
Mandelate with switched chirality
Comment on the sequence identity and the structural similarity of enzymes in the enolase family
- Where is conservation the highest
Very low sequence identity
Have very similar structures
- Conservation at its highest in the active sites; Particularly with metal ion binding residues
