Warren Flashcards
Specificity is imposed during:
Initial binding
Induced fit
Chemical steps of catalysis
Specificity constant
kcat/Km
Gives a measure of the catalytic efficiency
Preference for one substrate over another
6 types of enzymes
Oxidoreductases Transferases Hydrolase Lyases Isomerase Ligase
Two reasons why enzymes are so large
Flexibility- sufficient flexibility for active site
Rigidity- extract spatial array for activity
Co factors and co enzymes
2 examples of co enzymes
30% are metalloenzymes
Pyroxidal phosphate, active vb6. Covalently binds substrate, and acts as an electrophillic catalyst
Thiamine pyrophosphate- B1 derivative, catalyses reversible decarboxylation reactions e.g. Acid or alcohol
Thermodynamic lability
How easily the substrate is changed
Kinetic stability
How stable the compound is
E.g. Glucose on shelf
3 main ways of catalysing a reaction
Stabilising TS
Destabilising substrates
Replace single step with multi steps
How do enzymes lower activation?
Using the intrinsic binding energy to catalyse the reaction
The 4 types of enzyme catalysis
Approximation (entropic) Covalent catalysis (entropic) Acid base (enthalpic) Strain distortion (enthalpic)
Catalysis by approximation
Degrees of freedom
Close proximity
Example of imidazole catalysed p-nitro acetate
Draw it out
Entropic- since the probability of reaction is increased when they are bound in a specific orientation
Product loses degrees of freedom which is unfavourable
Binding stops rotational and translational freedom of substrates
This is paid for by the binding energy
Covalent catalysis
Covalent adducts- between active site and subtrate
Immobilisation- entropic driving force as the system wants to increase entropy
Accommodates multiple steps in a single active site
Used by catalytic triads e.g. Chymotrypsin
The cofactors TPP and PLP
Example of pyruvate dehydrogenase complex using TPP
General acid base catalysis
Enthalpic
Proton is transferred in transition state
Stabilises TS
Avoids formation of unstable species
Nucleophilicity of water increased without increasing OH-
Often His
Serine protease- His accepts H from Ser, allowing Ser to attack amide bond
Catalysis by strain
Enthalpic
Each bond has a binding energy
Strain in starting product and release of strain in the TS to products
Inducing strain lowers the intrinsic binding energy
Work done to move bond paid for by energy
Strain includes
Geometric distortion of bond angles
Steric compression
Electrostatic repulsion
Desolation of charged molecule in hydrophobic site
All lessen the energy barrier to TS
Destabilisation: tight binding ratio
More like TS, more tight binding
Less binding energy used for destabilisation
Proline racemase- planar analogue binds x160 tighter
Explains why TS analogues are competative inhibitors- all binding energy directed to tight binding and none to driver catalysis
Antibodies
Very low Kd, 10-10 M
Enzymes haven’t evolved to bind tightly or they won’t release products
Why aren’t enzymes perfect?
Must reflect substrate conc
Binding affinities similar to biological levels
Tight binding would compromise kcat/km
Diffusion controlled limit
If kcat > k-1 then a smaller Km would mean a slower turnover number
Non productive binding
Selectivity of good substrates How binding energy gives specificity Example of hexokinase Basicity of water and glucose OH similar Only glucose gives induced fit Binds in catalytically productive mode Water doesn't induce this change
Two types of chelatases
Insert metal ion to tetrapyrrole
1- 3 subunits, hydrolyse ATP (enzyme recording)
2- small, single unit, no ATP.
Type 2 chelatases
4 types
CbiK- sirohydrochlorin + Co2+ -> cobalt-sirohydrochlorin + 2H
Used in B12 synthesis
Also catalysed by CbiX
SirB- sirohydrochlorin + Fe -> sirohaeme
HemH- protoporphyrin IX + Fe -> Heme
Sometimes dimeric and membrane associated
Rough structure of chelatases
Bi-lobal
Metal and porphyrin binding site
Chelatases mechanism
Metal bind to 2 His (His and Glu in ferrochelatase)
His remove 2H from ring
Insertion of metal
Negatively charged channel to attract metal
Chelatases and the 4 types of catalysis
Approximation- metal and substrate close, compensates for degrees of freedom loss
Acid/base- use of His to remove protons. Likely to mimic transition state
Strain- pushes pyrrole ring out of plane. Makes central cavity larger and encourages metal binding. Return to planar causes expulsion.
Inhibitors of chelatases
N-methyl porphyrins inhibit ferrochelatase
Used to make antibodies with ferrochelatase activity
These mimic the transition state as the ring isn’t planar
Abzymes
Antibody to N-methylmesoporphyrin Antibody to transition state Stabilises Less effective than enzyme How does it recognise metal substrate?
Type I chelatases
Mg into chlorophyll. ChiH, I, D Cobalt into B12, CobN, S, T H or N is 140 KDa Other subunits form hexamer if complex E.g. (ID)6 20 ATP per insertion
Why use lactate dehydrogenase as a model?
Well studied
Crystal structure
Pyruvate + NADH -> lactate + NAD
Ordered reaction
Mechanism of lactate dehydrogenase
Transfer of H from enzyme and H from NADH to the =O of pyruvate
Release of lactate
Enzyme reprotonated on His before NAD release
His195 in LDH
pH titrations
Needs to be protonated for catalysis
Used diethylpyrocarbonate to inhibit His residues which stopped reaction
Arg109 LDH
Induces a dipole on carboxyl in acid of pyruvate
Stabilises transition state
R109Q mutation drops to 0.07% hydride transfer
And lowers pyruvate affinity to 5%
Asp168 LDH
D168N and D168A mutations (Clarke 1988)
Showed that 168 forms strong interaction with His195 by raising the pKa and anchoring orientation
Replacement shows
Both reduced affinity and kcat
No change in His PKA when change to neutral
So Asp only interacts with closed form
Arg171 LDH
Forked 2 point interaction with the acid group of pyruvate
R171K variants - one point, lowers binding affinity
Reduced to 0.05% of WT
Affect the orientation
Arg has greater hydration potential
More water is displaced by Arg
Greater entropic component in Arg bond
Ile250 and NADH binding LDH
Hydrophobic environment for NADH ring
I250N- binds NADH weaker, 0.1%
The Ile is near to Arg, providing a very non polar environment for the the guanidium group of Arg to allow it to bind the carboxyl group of pyruvate
Polar environment lowers binding affinity
3 factors considering in converting LDH to MDH
Overall charge balance in active site
Influence of substrate and active site volumes
Effect of direct electrons tic complementarity
1- balancing the charge
Delete negative charge in active site
D197N -> x25 improvement
E107Q -> x2 improvement
Shows that more effective when balanced in active site
2- removing bulk from active site
T246G
300 change in LDH:MDH activity
Stops being efficient for pyruvate, not increase in efficiency for OXA
3- effect of direct electrostatic complementarity
Direct protein counter ion Q102R Provides a positive charge for the extra COOh side chain 8400x increase Favour OAA
Manipulation of effector control
FBP has 2 negatively charged phosphates to allow it to form tetramers of the enzyme
R173Q means that the enzyme can form a tetramer in absence of effector/activator
What are BMCs?
Bacterial organelles
Protein capsid shell
100-150nm, 5-20k subunits
The carboxysome
Cyanobacteria
Efficiency of carbon fixation
Prevents contact of rubisco with oxygen
HCO3 imported and converted to CO2 by carbonic anhydrase
Protein she’ll stops co2 leaving and stops photorespiration
CsoS1 or Ccmk hexamer shapes connected at edge by Cso54 or Ccml
Centre of hexamers has transport pore
Cobalamin metabolism in bacteria
Enterobacteria show cobalamin dependent metabolism
Ethanol amine and 1,2- propanediol as carbon and energy sources
Metabolosome
Both are needed for survival of salmonella enterica in gut
Not sure why yet
The metabolosome
1,2 PD imported
PduCDE converts to propionaldehyde.
Even converted by propanol dehydrogenase -> propanol outside
Or
PduP breaks down to propionyl-CoA and creates NADH
Exits before propionate
The Pdu Operon
She’ll protein, enzymes
Same Operon
Used for propanediol system
Can redesign this with different proteins
BMC for ethanol production
5-6 genes for an empty shell
Proteins targeted by attachment to the normal BMC proteins
Peptide sequences from PduP and PduD
Target pyruvate de carboxylase and alcohol dehydrogenase
Pyruvate -> acetaldehyde -> ethanol
