Enzyme Mechanisms Flashcards
What are the three things reaction mechanisms determine?
Where electrons are
Which bonds are broken/made
Flow of electrons
Describe BrØnsted acids and bases
Acid- proton donor
Base- proton acceptor
Describe lewis acids and bases
Acid- can form coordinate bonds with lone pairs
Base- has lone pairs available for sharing
What is the pH equation with a certain pKa?
pH = pKa + log10([A-]/[HA])
What factors determine acidity of an organic compound?
Strength of Y-H bond
Electronegativity of Y
Factors to stabilise Y- compared to YH
Nature of the solvent
What does pKa depend on?
Temperature
Ionic strength of solvent
Microenvironment
Describe the protonated and deprotonated forms of histidine
The protonated imidazolium cation is stabilised by 2 resonance structures
Deprotonated imidazole has 2 tautomers where either N is protonated
Why is cysteine more acidic than serine?
The S valance electrons are further from the nucleus than in O, so electrons are easier to donate
Describe RNase structure
V shaped
3-stranded antiparallel β-sheets
N-terminal helix with His12
4 SS bridges
What do RNases cut?
They act as endonucleases on ssRNA
Cleave P-O5’ bond
Cuts after a pyrimidine base (U or C), suggesting there must be a recognition site
Describe the key catalytic intermediate of the RNase reaction
2’-3’ cyclic nucleotide. This is cleaved in the second step of the RNase reaction
How were kinetic studies used to predict the active site of RNase?
The pH profile of Vmax is around 6-7, suggesting there might be 2 His residues in the active site- one acid, one base
How were His12 and His119 identified as active site residues in RNase?
Modified RNase could only have one key His modified at a time, but not together, His119 was 88% and His12 12% of the time, so one acted as an acid, the other a base
Describe the RNase A active site residues
His12 and His119 are directly involved in cleavage
Lys41 stabilises negatively charged phosphates in the intermediate
Describe the RNase A specificity pocket residues
Phe120- van der waals contacts with the RNA base
Ser123 and Thr45 are involved in hydrogen bonding
The pocket is too small for purines (AG)
What is the acidic active site residue of RNase?
His12
Give an example of a serine protease
Trypsin
Chymotrypsin
Elastase
Thrombin
Kallikrein
Compliment System
Blood clotting factors
What sort of reaction do serine proteases catalyse?
Hydrolysis of a scissile bond with water
How do the S1 and S1’ pockets bind to P1 and P1’ respectively?
S1 binds upstream and S1’ downstream of the scissile bond
How is chymotrypsin initially synthesized?
Inactive enzyme precursor (zymogen) called chymotrypsinogen
Where about in a polypeptide does chymotrypsin cleave?
After large aromatic residues (Phe, Tyr, Trp)
What is the catalytic triad in the chymotrypsin active site?
Ser195
His57
Asp102
How can Ser195 be modified in chymotrypsin?
PMSF blocks serine proteases by modifying Ser195, inhibiting the enzyme
DIPF is a nerve gas which blocks serine proteases and related molecules
How can His57 in Chymotrypsin be identified?
TPCK is a substrate analogue which binds at the active site and is nucleophilic attacked by His
Describe trypsin-like serine protease structure
Two β-barrels formed from 6 anti-parallel β-strands
His57, Ser195, Asp102 catalytic triad
Oxyanion hole
Specificity pocket
Main chain substrate binding
Describe how the chymotrypsin catalytic triad is stabilised
-ve Asp102 stabilises formation of +ve His57, so the proton from Ser195 can donate to His57
This makes Ser195 nucleophilic
Describe the chymotypsin oxyanion hole
Located near the carbonyl of the substrate scissile bond
Backbone amide hydrogens of Ser195 and Gly193 point to the active site to stabilize the tetrahedral enzyme-substrate intermediate
Describe the chymotrypsin mechanism
- His57 attacks Ser195 which attacks the carbonyl group of the substrate, forming a covalent bond. This forms a -ve tetrahedral intermediate stabilised by the oxyanion hole
- Tetrahedral intermediate decomposes to the acyl-enzyme intermediate with His57 donating the proton
- The enzyme is deacylated through another tetrahedral intermediate. Water is the nucleophile, Ser195 is the leaving group
What % identity do chymotrypsin and trypsin have?
40%
What residues are found in the chymotrypsin, trypsin and elastase specificity pockets?
Chymotrypsin: G266, S189, G216
Trypsin: G266, D189, G216
Elastase: V226, T216
How do serine proteases orient polypeptides for cleavage?
Bind to the side chains in the specificity pocket and recognise the main chain conformation.
How are serine proteases controlled?
Produced as zymogens (proenzymes) which require proteolytic cleavage of their precursors to activate
What is the protease activation cascade master activator?
Eteropetidase
how is α-chymotrypsin activated?
- Chymotrypsinogen is partially activated by cleavage by trypsin to π-chymotrypsin.
- This activates other π-chymotrypsin molecules to form α-chymotrypsin.
- The A,B and C chain are connected by disulfide bonds
How is the chymotrypsin active site activated?
The N-terminal Ile16 on π-chymotrypsin ionically pairs to the Asp194 side chain.
This changes the position of the Ser195 side chain for the correct geometry.
The amides of residues 193 and 195 are oriented to form the oxyanion hole
Where does thrombin cleave fibrinogen?
Arg-Gly
This forms plaques of insoluble fibrin, forming clots
How are serine proteases used in the blood clotting cascade?
Thrombin cleaves fibrinogen to form insoluble fibrin
Positive and negative feedback form the activation and deactivation systems
Plasmin/plasminogen system dissolves clots
Give an example of convergent evolution of serine proteases
Subtilisin is a bacterial protease with an unrelated sequence to chymotrypsin. However it has the Asp, His, Ser catalytic triad
Describe proteasomes
Intracellular complexes with proteolytically active sites in its interior cave belonging to N-terminal threonine hydrolases.
4 stacked rings of 7-fold symmetry
What sort of activity do the protease active sites of the inner (β) subunits of the proteasome have?
β1- caspase
β2- tryptic
β5- chymotryptic
How does bortezomic inhibit apoptosis?
Induces the ubiquitin-proteasome pathway by binding to the β5 subunit.
It has a trigonal conformation which mimics a peptide bond.
When boron is attacked by a nucleophile, it is converted from trigonal sp2 to tetrahedral sp3.
What cellular processes are associated with cysteine proteases?
Apoptosis, parasitic infections, virus maturation
Outline the action of a cysteine protease
- Nucleophilic attack by a thiolate on C=O
- Tetrahedral intermediate forms in the oxyanion hole
- A water molecule is activated by His and attacks the carbonyl group
- Second tetrahedral intermediate forms
What is Burkoldria?
B. pseudomallei. Potential bioterror weapon.
Multi-drug resistant bacterium with no vaccine.
Infection mimics TB
7 morphotypes with different patterns of gene expression
How does BPSL1549 from burkholdria compare to the CNF1 domain?
They have structural, but not sequence similarity. This is an example of convergent evolution.
The catalytic His-Cys pair is conserved
Describe CNF1
114kDa pathogenic toxin expressed by some E.coli
Deamidates key glutamine residues in small GTPases (Rho, Rac and Cdc42)
Inhibits GTP hydrolysis, by deactivating GTPases. This leads to cytoskeleton remodelling
Describe BPSL1549
23kDa protein in Burkholdria.
Unknown function and no sequence similarity to proteins outside of burkholderia.
Deamidates Gln339 in RNA helicase eIF4A, preventing initiation-dependant protein translation.
What metal ion facilitates catalysis of the enolase superfamily?
Mg2+
Outline the mandelate racemase mechanism
Lys166 acts as a base to remove H from the chiral carbon.
The OH then attachs the carbonyl C=O
His297 acts as an acid and returns the proton
Describe the mandelate racemase active site
The Mg ion is coordinated by carboxylic acid groups on Asp/Glu
Mg2+ stabilises the acid-carboxylate
The L-proton faces Lys166 and catalytic histidine returns the D-proton on the opposite side
What is a common H- source in lab settings
BH4- (Borohydride)
Why are biological reductants better than BH4-?
They use cofactors which are directed by enzymes to target molecules, making them very specific
Describe the main features of an NAD ring
Net positive charge all over
Phosphate backbone in nucleotides gives an overall negative charge
Why is hydride transfer very stereospecific?
The enzyme differentiates between hydrogens pro-R/pro-S on the carbon.
Hydride transfers to a particular face of the nicotinamide ring
What parameters are critical to hydride transfer?
Distance and angle of approach of hydride.
In NADH, this is 3-3.5Å, also known as van der Waals distance
What are the basic steps in fatty acid biosynthesis?
Oxidation -> hydration -> oxidation
Describe type I fatty acid synthase
Contain catalytic domains 1 and 2
Found in bacteria, vertebrates and yeast
Describe type II fatty acid synthase
Contain discrete polypeptides catalysing each enzymatic step
Found in plants and many bacteria
How do acyl carrier proteins bring acyl groups to fatty acid synthase?
Its phophoantetheine covalently linked to a serine residue ( S-acyl primed) attacks the acyl group.
What is the first step of fatty acid elongation?
Oxidation: uses β-keto ACP reductase
NADP+ reduced
tetrameric to trigonal planar
Describe the β-keto ACP reductase (BKR) active site?
Rossman fold and S…YxxxK motif.
Tyr and Lys in the active site
Describe Bassica napus ENR (enoyl-acyl carrier protein reductase)
Isoform from developing rapeseed seeds
Isolated from chloroplast
Tetramer with each subunit being 32.5kDa
NADH specific
How do the structures of BKR and ENR compare?
High structural similarity despite <20% sequence identity
Active site tyrosines do not directly superimpose
Phenyl hydroxyl groups are very close in position
Outline the ENR mechanism
Hydride transfer from NADH to C3 at a double bond in the acyl substrate
Rearrangement forms an enolate anion
A proton is donated from tyrosine side chain
Tautomerisation from Enol to keto to give a reduced product
How is the ENR active site postitioned?
Positioned for the nicotinamide ring to go in
What does a G93 to V93 change in enolate reductases (ENR) cause?
Not much change as van der waals forces overlap
How can phosphodiester bond hydrolysis be described?
SN2:
Substitution
Nucleophilic
2 molecules (bimolecular)
What is the associative mechanism of phosphodiester bond cleavage?
Water attacks phosphate
HOR group leaves
Trigonal pyramidal intermediate
What is the dissociative mechanism of phosphodiester bond cleavage?
Water molecule sits close to the phosphate
Trigonal planar intermediate forms
OR group leaves
How can RNA phosphodiester bonds self-cleave?
Alkaline conditions
Base attacks 2’ OH on ribose which attacks 3’ end of phosphate
What are the key parts of RNA nucleases?
His12 cleaves phosphate group
His119 accepts the proton
