Enzymes Flashcards
Enzyme-substrate complementarity
Substrate and residues in active site as close together as possible for maximum binding energy (snug fit)
Greater complementarity + greater binding energy
Factors involved in binding substrate
H bonds
Electrostatic
Hydrophobic
VDW
What is Kcat
Turnover number
What is Kcat/Km
Indicated whether an enzyme-catalysed reaction is diffusion controlled
I.e. chemical+release steps are so fast that enzyme is just waiting to encounter substrate
10^8 to 10^9 M-1S-1
Transition state theory
Transition state represents the most unstable species in a reaction pathway where chemical bonds are being broken and made and is located at the peak of the reaction profile
Intermediates have fully formed bonds and occupy troughs in the profile
Kcat/Kuncat = e^change in binding energy/RT
Active site of an enzyme has evolved to bind TS more tightly than the substrate
Gives maximum binding energy
General acid catalysis
Process whose rate is dependent on the concentration of all acids present in the reaction not just {protons] of aqueous reactions, e.g. a side chains
General base catalysis
Process whose rate is dependent on the concentration of all bases present in the reaction, not just [OH-] if aqueous reaction
Specific acid / base catalysis
Reactions whose reactions are just dependent on [H+] or [OH-]
Concerted general acid-base catalysed reaction
Both processes occur simultaneously, only one TS, proton delivered and accepted at same time
PH dependency of acid-base catalysed reactions
Reversible reaction where aa side chains are deprotonated
Above pKa= deprotonated
Must be in correct protonation state for enzyme to be active
Effect of environment on pKa values
PKa values can be shifted by micro environment
Polarity + presence of charges
Hydrophobic region = uncharged species favoured = increase pKa
+ve charge nearby = -ve charge stabilised = decreased pKa
-ve charge nearby = -ve charge destabilised = increased PKa
Covalent catalysis
Formation of covalently bound enzyme intermediate
Form from reaction of the substrate with an aa side chain or a coenzyme
Aa side chains can act as nucleophiles but only reactive when deprotonated
Important in group transfer reactions - 2 substrate reactions that usually involve transfer of one group from one substrate to another
Important in making substrate more reactive
E.g. formation of schifi bases to activate carbonyl groups
chemical trapping of enzyme intermediates
React enzyme with NaBH4, source of hydride ions
Forms amine, non-reactive , prevents further catalysis
Enzyme inactivated as modified form of substrate is permanently attached to Lys side chain
Electrostatic catalysis
Presence of charges and oriented dipoles within the active site can stabilise TS by solvation
TS accumulates charge, surround TS with opposite charge to get favourable charge-charge interactions to get binding energy
Coulombic interactions are more effective in non-polar environments as they have lower dielectric constant
Metal ion catalysis
Metalloenzymes containing tightly bound ions e.g Fe2+ or Mg2+
Metal-activated enzymes loosely bind ions from solution e.g. Na+ , K+
What are metal ions involved in
Electrostatic stabilisation and charge screening
Highly effective because of high charge that is pH independent
E.g. Mg2+-ATP complex in kinases
Redox reactions
Binding and orientation of substrates
Act as Lewis acids and can form dative or covalent coordinate bonds
Promote nucleophilic catalysis by activating water bound molecules
Catalysis through orientation and proximity effects
Rate enhancement obtained by taking 2 reactants out of solution and placing them next to each other in the enzyme, raising the local concentration of each reactant
Binds substrate in precise geometry so orbitals have correct orientation
Entropy changes in bimolecular reactions
A + B —> A-B has large -ve delta s
A + B —> AB in enzyme —> A-B smaller -ve S so more favourable
Enzyme can tether reactants together to increase local conc
Serine protease catalytic triad
Asp-102
His-57
Ser-195
Serine protease mechanism
1)Nucleophilic attack of serine to form first tetrahedral intermediate
His-57 general base
Asp-102 electrostatic effect
2) decomposition of first tetrahedral intermediate to give Acyl-enzyme intermediate
His-57 general acid to form amine leaving group
3) His-57 acting as general base promotes nucleophilic attack by water on the Acyl-enzyme to form second tetrahedral intermediate
4) decomposition of intermediate to give resting enzyme and carboxylic acid
Protonated his-57 acts as a general acid
Serine protease oxyanion hole
Acts to stabilise transition state
In michaelis complex, trigonal carbon of scissile peptide bond is conformationally constrained from binding into oxyanion hole
In tetrahedral intermediate, the oxyanion species enters the hole and makes favourable H bonds to the backbone NH groups of Gly193 and ser 195
An additional H bond to gly193 is also made
Favourable binding interactions reduce energy of activation
Carbonyl oxygen shifts into cavity
Charged group stabilised by residues in enzyme
Only occurs in TS
Specificity of serine proteases
Determined by nature of S1 pocket and p1 residues of substrate
Chymotrypsin cleaves bulky P1, Phe, trp, Tyr
Trypsin has asp in pocket so cleaves +ve P1, Arg, Lys
Elastase has 2 valines in pocket s cleaves small, neutral P1, Ala, gly, ser, val
Divergent vs. Convergent evolution
Divergent is after a gene duplication event
Only need one copy of the gene so now second can mutate without causing harm
Produced trypsin, chymotrypsin, elastase
Convergent evolution produces same triad but with completely separate route
Kinetics of chymotrypsin
Shown with pre-steady state kinetics
Fast initial burst of product to generate covalently attached Acyl-enzyme intermediate
Slower regeneration of enzyme is RDS of catalytic cycle and limits catalysis in subsequent turnovers
Cysteine proteases
Similar to serine
Use thiol group which has lower pKa than ser
No aspartate as it doesnt need a base to pull off the proton
Aspartyl proteases
Water attacks directly
Aspartate in conjugate base form to pull off a proton
Second asp in protonated form ti H bond with oxyanion species to stabilise it
2 asp residues must have different pKa values
Metallo proteases
Similar to carbonic anhydrase
Tightly bound metal ion can bind water
Appropriately positions H2O to attack
Base close by to pull off proton
Electrostatic interaction between Zn2+ and oxyanion
Hydrolysis of peptide bond is favoured by
Presence of nucleophile to attack carbonyl
Presence of charges to polarise carbonyl and stabilise tetrahedral intermediates
Presence of proton donor to make -NH a better leaving group
How is trypsinogen activated
Serine proteases are synthesised as larger precursor molecules called zymogens in pancreas that show little activity
Trypsinogen activated by cleavage after lys15
Initially cleavage is catalysed by a serine protease under hormonal control
Trypsin can then catalyse it’s own activation (autocatalytic)
Inappropriate activation in the pancreas is prevented by synthesis of pancreatic trypsin inhibitor which binds tightly to trypsin
Proelastase is activated in a similar manner
Activation of chymotrypsinogen
Trypsin activates chymotrypsinogen by removing dipeptides
Ser14 Arg15 and Thr147 Asn148
The protein is linked by disulphide bridges
What does lysozyme do
Destroyed bacterial cell walls by hydrolysing the beta 1-4 glycosidic linkage between NAM and NAG sugar units
Groove of lysozyme
6 sugar units a-f fit into groove of enzyme
Cleavage between e and d
All 6 units needed to drive catalysis
Binding energies of all units apart from d are positive
D has a high, unfavourable binding every
Explains why you need more sugar as you need favourable binding to drive unfavourable binding
Binding to sub site d involves structural distortion (strain) of the sugar
Sugar in subiste d adopts half chair conformation
Philips mechanism for lysozyme
SN1-like
1) glu35 acts as a general acid and protonates bridge oxygen of the glycosidic bond
Unusually high pKa to ensure in protonated state
2)glycosidic bond cleaves, leaving a positively charges D ring Oxonium ion which is stabilised by favourable electrostatic interaction with -ve asp52 and the enzyme induced distortion of the D ring to enhance resonance stabilisation
3) glu35 in deprotonated state acts as a general base to activate water to perform addition reaction at carbocation
Lysozyme koshland mechanism
2 back to back sn2-like steps
1) -ve asp52 acts as a nucleophile to displace the first product
In a concerted mechanism, glu35 acts as a general acid and protonated bridge oxygen of the glycosidic bond
Glu35 has an unusually high PKa to ensure in protonated state and reaction aided by distortion of the d ring
2) Glycosyl-enzyme intermediate is formed
3) glu35 in deprotonated state acts as a general base to activate water to perform nucleophilic sn2 reaction to displace asp52 to release the second product
*detection of the Glycosyl intermediate suggests this mechanism operates
Hexokinase
First step of glycolysis
Sn2 reaction
Phosphoryl group is transferred into oxygen
Competing reaction is H2O hydrolysing atp
Mechanism of Hexokinase
Penetrate -ve charge barrier on p by forming dative covalent bonds with mg2+ to give neutral species
Nucleophilic attack of the c6-oh group of glucose onto the gamma phosphate of an mg2+-atp complex (metal ion catalysis)
Hexokinase is an example of induced fit
Movement of lobes upon substrate binding squeezes out water, preventing the possible hydrolysis of atp in competing side reaction
Glucose partially buried
Lactate dehydrogenase role
Catalyses stereospecific formation of lactate from pyruvate
In principle reaction could form 2 enantiomers
Only L isomer is formed
In the hydride reduction of a carbonyl, hydride can attack from above or below the plane
R and S
R stereoisomer is 1,2,3, clockwise
S stereoisomer is 1,2,3, anticlockwise
Same logic for re and si
Pro r and pro s of NADH
C4 is prochiral
Mechanism of lactate dehydrogenase
Reactants are pinned in precise geometry in the enzyme by Arg resides forming hydrogen bonds
Only the pro-r hydrogen is transferred and always to the re face of pyruvate
An example of absolute Stereospecificity
Geometric and electronic complementarity in the michaelis complex confers substrate specificity
Allosteric activator
Stabilises relaxed form
Allosteric inhibitor
Stabilises tense form
ATCase role
Catalyses committed step in pyrimidine biosynthesis
Carbamoyl phosphate + aspartate —> N-carbamoyl Aspartate
ATCase structure
3 regulatory dimers
2 catalytic trimers
Zinc motif binds subunits
Active site contains residues from more than one subunit
Side chains contribute to specific binding
Each binding site is a combination of 2 subunits
Control of ATCase
Pala is competitive inhibitor
Also controlled by feedback inhibition
As [CTP] increases, rate of N-carbamoyl aspartate formation decreases
R state favoured by substrate binding
T state favoured by CTP binding
CTP is negative regulator
ATP is positive regulator
Sigmoidal kinetics of ATCase
Active sites cooperate
Titration behaviour when substrate is added
Cooperativity- binding of ligand at one active site affects binding at other sites
Model for cooperativity is 2 michaelis menten enzymes with different km values
Key MWC parameters
L = [T0]/[R0]
Larger L means t to R equilibrium lies further towards t state
C = kR/kT
Smaller value of c means greater affinity of the substrate for R state compared to T state. Higher c means smaller difference in binding between t and r forms so less of a cooperative transition
N
Higher value n = more substrate can bind = T-R equilibrium can be shifted more towards R state. Smaller n means less cooperative effect
Structure of Haemoglobin and myoglobin
Mb is monomeric and stores o2 in tissues
Hb is an alpha2beta2 tetramer and carries o2 in blood
Mb has single O2 binding site
Ligation oh haem
Haem has 6 potential ligand positions
4 are occupied by planar porphyrin ring nitrogen’s
1 ligated by proximal histidine
In absence of o2, Fe is below the plane of the ring
When o2 binds, d orbitals contribute to bonds so VDW radius shrinks and Fe fits into plane of ring
Protein responds to motion as proximal histidine is pulled up by 0.4A
Resonance structures of bound O2
Fe2+—O=O dioxygen
Fe3+—O—O- superoxide (deadly for cells)
ferrous(Fe2+) is stabilised in Mb by distal histidine
Structure of Haemoglobin prevents oxidation of Fe
Quantification of ligand binding to single independent site
KD = [P][L]/[PL]
[PT]=[P]+[PL]
KD = [L] that gives 50% binding
Cooperativity of Hb binding
Sigmoidal curve
Hb has 4 binding sites
2 pairs of alpha beta dimers
Alpha1beta1 and alpha2beta2
Strong interactions between alpha1beta1 and alpha2beta2 interfaces
Strong interactions between alpha and beta
Alpha helices lean against each other and push and pull
Hill coefficient
Measure of cooperativity
If n>1 it suggests 2 or more binding sites and +ve cooperativity
<1 is negative cooperativity
1= no cooperativity
At extreme values, Hb is mostly T state, small changes in O2 pressure wont produce much R
When almost saturated any changes in occupation wont produce more R as hardly any T left
What causes T to R transition in Haemoglobin
The fe2+ atom moves into the plane of the haem upon oxygen binding
Helix containing his ligand moves
Movement of the helix alters the interface between the alpha beta pairs
Alpha beta pairs slide and rotate upon formation of the R state
Allosteric regulation of Hb
Negative regulators promote O2 release
2,3-bisphosphoglycerate (produced in RBC glycolysis)
CO2
H+
How does 2,3-BPG promote oxygen release from Hb
Stabilises T form
BPG occupies central hole in t form
Increases BPG levels allow acclimation to high altitude
More bpg shifts binding curve to the right, less Hb occupied
Foetal Haemoglobin has alpha2gamma2 form with reduced affinity for BPG
His 143 mutated to serine
Still good binding interactions
Binding curve shifts left as r state stabilised so O2 flows into blood circulation foetus
PH and CO2 in oxygen release
Decreasing pH (Bohr effect) and increasing co2 levels promotes O2 release
Co2 is a weak acid so creates protons, causing binding curve to shift right as t state stabilised
Protonation of his146 by lowering pH promotes stabilisation of T form of deoxyhaemoglobin
Transport of CO2 out of body
CO2 reacts with N terminal amino groups to generate carbamate
Carbamate transports CO2 out of body attached to Hb
Most CO2 transported through body dissolved in blood
Most transported to lungs in form of bicarbonate produced in RBS and then released into blood plasma
What is a cofactor
Organic molecules/ions required by an enzyme for activity
What is holoenzyme and apoenzyme
Holoenzyme is cofactor + protein
Apoenzyme is protein alone (inactive)
What is a vitamin
Small organic molecules that are essential for growth but unable to be synthesised by the organism so must be present in the diet
What is biotin
Has an imidazoline ring that is cis-fused to a tetrahydrothiophene ring with a valerete side chain
Valerete side chain holds biotin into active site as prosthetic group
Covalently attached to enzyme
In the carboxybiotinyl enzyme, N1 of the biotin ureido group is in the Carboxylation site
Valerete side chain has amide link to lysine side chain in enzyme
Biotin binds co2 and delivers it to relevant active site
Role of biotin in pyruvate carboxylase
Biotin moves co2 huge distances
Pyruvate carboxylase has 2 subunits
1 domain for Carboxylation
2nd domain for carboxyltransferase
3rd domain with biotin attached on swinging arm
Swinging arm swings between 2 subunits
Connects 1st domain of one subunit with second domain of the other
What is pyridoxal phosphate (PLP)
Has reactive aldehyde group and acidic phenolic group
6 member ring with a nitrogen
N lone pair not delocalised in ring so n can act as a base
Reactive aldehyde reacts with amino groups to form schiff base
Acidic phenol has lower pKa so can be deprotonated
Can H bond to intermediates or play role in acid-base catalysis
Forms tautomer when H from phenol moves onto N
NH+ can then act as temporary e- sink to stabilise carbanions
Stable tautomeric form allows PLP to act as an e- sink during reactions
Aminotransferase reaction
Aa1 + enzyme-PLP —> alpha keto acid 1 + enzyme-PMP
Amine is transferred from aa1 to pyridoxal group
Transfer of amino acid is covalent catalysis
PMP shows it has attached amino group
Alpha keto acid 2 + enzyme-PMP —> aa2 + enzyme-PLP
Amine transferred from pyridoxamine group to alpha keto acid 2
Group transfer reaction is via ping pong mechanism
How is amino group transferred in aminotransferase reaction
Transferred by tautomerisation of schiff base
Proton needs to be weakly acidic
Pyridine ring stabilises carbanion
Proton is detached and moved to another carbon because of octet rules the double bond has to shift
Mechanism of aminotransferase
Transamination
Tautomerisation
Hydrolysis
How is aminotransferase intermediate stabilised
Planarity is important for resonance stabilisation
Essential for pi orbital overlap
Configuration of the intermediate is tightly controlled by the architecture of the enzyme active site
Carboxylate of alpha carbon is held in place by coordination to an Arg residue
H bond keeps system planar
The proton that is being transferred can only come from one direction so only one stereoisomer produced
All aas have 2S configuration apart from Gly and Cys
Elimination occurs perpendicular to the plane of the delocalised system
Rotation around C-N bond brings different groups perpendicular to delocalised pi system
When bond is broken C forms p orbital that can delocalise into system
