Warren

Question 1

Specificity is imposed during:

Answer 1

Initial binding
Induced fit
Chemical steps of catalysis

Question 2

Specificity constant

Answer 2

kcat/Km
Gives a measure of the catalytic efficiency
Preference for one substrate over another

Question 3

6 types of enzymes

Answer 3

Oxidoreductases
Transferases
Hydrolase
Lyases
Isomerase
Ligase

Question 4

Two reasons why enzymes are so large

Answer 4

Flexibility- sufficient flexibility for active site

Rigidity- extract spatial array for activity

Question 5

Co factors and co enzymes

2 examples of co enzymes

Answer 5

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

Question 6

Thermodynamic lability

Answer 6

How easily the substrate is changed

Question 7

Kinetic stability

Answer 7

How stable the compound is

E.g. Glucose on shelf

Question 8

3 main ways of catalysing a reaction

Answer 8

Stabilising TS
Destabilising substrates
Replace single step with multi steps

Question 9

How do enzymes lower activation?

Answer 9

Using the intrinsic binding energy to catalyse the reaction

Question 10

The 4 types of enzyme catalysis

Answer 10

Approximation (entropic)
Covalent catalysis (entropic)
Acid base (enthalpic)
Strain distortion (enthalpic)

Question 11

Catalysis by approximation

Degrees of freedom

Answer 11

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

Question 12

Covalent catalysis

Answer 12

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

Question 13

General acid base catalysis

Answer 13

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

Question 14

Catalysis by strain

Answer 14

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

Question 15

Destabilisation: tight binding ratio

Answer 15

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

Question 16

Antibodies

Answer 16

Very low Kd, 10-10 M

Enzymes haven’t evolved to bind tightly or they won’t release products

Question 17

Why aren’t enzymes perfect?

Answer 17

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

Question 18

Non productive binding

Answer 18

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

Question 19

Two types of chelatases

Answer 19

Insert metal ion to tetrapyrrole

1- 3 subunits, hydrolyse ATP (enzyme recording)
2- small, single unit, no ATP.

Question 20

Type 2 chelatases

4 types

Answer 20

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

Question 21

Rough structure of chelatases

Answer 21

Bi-lobal

Metal and porphyrin binding site

Question 22

Chelatases mechanism

Answer 22

Metal bind to 2 His (His and Glu in ferrochelatase)
His remove 2H from ring
Insertion of metal

Negatively charged channel to attract metal

Question 23

Chelatases and the 4 types of catalysis

Answer 23

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.

Question 24

Inhibitors of chelatases

Answer 24

N-methyl porphyrins inhibit ferrochelatase
Used to make antibodies with ferrochelatase activity
These mimic the transition state as the ring isn’t planar

Question 25

Abzymes

Answer 25

``` Antibody to N-methylmesoporphyrin Antibody to transition state Stabilises Less effective than enzyme How does it recognise metal substrate? ```

Question 26

Type I chelatases

Answer 26

``` 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 ```

Question 27

Why use lactate dehydrogenase as a model?

Answer 27

Well studied Crystal structure Pyruvate + NADH -> lactate + NAD Ordered reaction

Question 28

Mechanism of lactate dehydrogenase

Answer 28

Transfer of H from enzyme and H from NADH to the =O of pyruvate Release of lactate Enzyme reprotonated on His before NAD release

Question 29

His195 in LDH

Answer 29

pH titrations Needs to be protonated for catalysis Used diethylpyrocarbonate to inhibit His residues which stopped reaction

Question 30

Arg109 LDH

Answer 30

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%

Question 31

Asp168 LDH

Answer 31

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

Question 32

Arg171 LDH

Answer 32

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

Question 33

Ile250 and NADH binding LDH

Answer 33

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

Question 34

3 factors considering in converting LDH to MDH

Answer 34

Overall charge balance in active site Influence of substrate and active site volumes Effect of direct electrons tic complementarity

Question 35

1- balancing the charge

Answer 35

Delete negative charge in active site D197N -> x25 improvement E107Q -> x2 improvement Shows that more effective when balanced in active site

Question 36

2- removing bulk from active site

Answer 36

T246G 300 change in LDH:MDH activity Stops being efficient for pyruvate, not increase in efficiency for OXA

Question 37

3- effect of direct electrostatic complementarity

Answer 37

``` Direct protein counter ion Q102R Provides a positive charge for the extra COOh side chain 8400x increase Favour OAA ```

Question 38

Manipulation of effector control

Answer 38

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

Question 39

What are BMCs?

Answer 39

Bacterial organelles Protein capsid shell 100-150nm, 5-20k subunits

Question 40

The carboxysome

Answer 40

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

Question 41

Cobalamin metabolism in bacteria

Answer 41

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

Question 42

The metabolosome

Answer 42

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

Question 43

The Pdu Operon

Answer 43

She'll protein, enzymes Same Operon Used for propanediol system Can redesign this with different proteins

Question 44

BMC for ethanol production

Answer 44

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