Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Proteins > Warren > Flashcards

Warren Flashcards

1
Q

Specificity is imposed during:

A

Initial binding
Induced fit
Chemical steps of catalysis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Specificity constant

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

6 types of enzymes

A 
Oxidoreductases
Transferases
Hydrolase
Lyases
Isomerase
Ligase
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Two reasons why enzymes are so large

A

Flexibility- sufficient flexibility for active site

Rigidity- extract spatial array for activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Co factors and co enzymes

2 examples of co enzymes

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Thermodynamic lability

A

How easily the substrate is changed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Kinetic stability

A

How stable the compound is

E.g. Glucose on shelf

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

3 main ways of catalysing a reaction

A

Stabilising TS
Destabilising substrates
Replace single step with multi steps

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How do enzymes lower activation?

A

Using the intrinsic binding energy to catalyse the reaction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

The 4 types of enzyme catalysis

A 
Approximation (entropic)
Covalent catalysis (entropic)
Acid base (enthalpic)
Strain distortion (enthalpic)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Catalysis by approximation

Degrees of freedom

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Covalent catalysis

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

General acid base catalysis

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Catalysis by strain

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Destabilisation: tight binding ratio

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Antibodies

A

Very low Kd, 10-10 M

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why aren’t enzymes perfect?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Non productive binding

A 
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
19
Q

Two types of chelatases

A

Insert metal ion to tetrapyrrole

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

20
Q

Type 2 chelatases

4 types

A

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

21
Q

Rough structure of chelatases

A

Bi-lobal

Metal and porphyrin binding site

22
Q

Chelatases mechanism

A

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

Negatively charged channel to attract metal

23
Q

Chelatases and the 4 types of catalysis

A

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.

24
Q

Inhibitors of chelatases

A

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

25
Q

Abzymes

A 
Antibody to N-methylmesoporphyrin
Antibody to transition state
Stabilises
Less effective than enzyme
How does it recognise metal substrate?
26
Q

Type I chelatases

A 
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
27
Q

Why use lactate dehydrogenase as a model?

A

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

28
Q

Mechanism of lactate dehydrogenase

A

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

29
Q

His195 in LDH

A

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

30
Q

Arg109 LDH

A

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%

31
Q

Asp168 LDH

A

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

32
Q

Arg171 LDH

A

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

33
Q

Ile250 and NADH binding LDH

A

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

34
Q

3 factors considering in converting LDH to MDH

A

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

35
Q

1- balancing the charge

A

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

36
Q

2- removing bulk from active site

A

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

37
Q

3- effect of direct electrostatic complementarity

A 
Direct protein counter ion 
Q102R
Provides a positive charge for the extra COOh side chain
8400x increase
Favour OAA
38
Q

Manipulation of effector control

A

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

39
Q

What are BMCs?

A

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

40
Q

The carboxysome

A

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

41
Q

Cobalamin metabolism in bacteria

A

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

42
Q

The metabolosome

A

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

43
Q

The Pdu Operon

A

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

44
Q

BMC for ethanol production

A

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

Proteins (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions