Enzymes and their Mechanisms Flashcards by Daniel Goff

Acid and base definition

Acid - Proton donor
Base - Proton acceptor

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Rearranged Henderson-Hasselbach equation to compare ratio of [HA] to [A-]

pH - pKa= log10([A-]/[HA])

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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

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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

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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

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Tautomer vs Resonance structure?

Tautomer - Atoms move within structure
Resonance structure - Atoms don’t move, only electrons move

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What is acid-base catalysis?

When a proton is transferred to or from the transition state

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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

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What are the 2 steps of the RNAse reaction?

Formation of 2’-3’ cyclic intermediate
Cleavage of cyclic intermediate into final compound

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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)

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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

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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

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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

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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

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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

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What is the scissile bond?

Bond at which the hydrolysis cleavage occurs

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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