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Enzymes Flashcards

1
Q

What are the different catalytic mechanisms?

A 
Proximity
Orientation
Acid/Base
Nucleophilic
Strain
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2
Q

How does proximity induce catalysis?

A

increases local concentration

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3
Q

What examples can be used to illustrate proximity?

A

Rate of intramolecular phenyl ester hydrolysis slows as chain length increases
Five membered ring is favoured

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4
Q

What effects does orientation have on catalysis?

A

Restricts rotation and position to favour transition state

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5
Q

What is an example of orientation?

A

100 fold reduction in rate of PLP catalysed amino acid reduction upon 10’ rotation
Nucleophile, Central carbon and leaving group are colinear

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6
Q

What range of pH is required for stable proteins?

A

5-9

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7
Q

How can pKa of sidechains vary?

A

4-10

based on environment

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8
Q

Why is Histidine unusual in chymotrypsin?

A

Perfect pKa to act as an acid or a base dependant on enviroment

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9
Q

Which enzymes use acid/base catalysis?

A

Lysozyme Glu35
Chymotrypsin His
Ketosteriod isomerase: Asp38 base Tyr14 acid

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10
Q

Why does lysozyme Glu35 have a higher pKa?

A

Hydrophobic environment

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11
Q

What mechanism does acetoacetate decarboxylase use?

A

Nucleophilic attack from deprotonated Lysine to form schiff base

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12
Q

What factors are important for enzymes using nucleophilic attack mechanisms?

A

Proximity
Orientation
Water exclusion to induce polarity

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13
Q

Which residues can be used in nucelophilic attack?

A

Thiols, mostly Cysteine

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14
Q

How does strain induce catalysis?

A

Causing a structure resembling molecular and electronic transition state to reduce energy change

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15
Q

What mechanism does carboxypeptidase A use?

A

Zn ion to distort linear peptide to tetrahedral
reduces electron overlap
carbonyl acts more like ketone

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16
Q

Why are cofactors used?

A

increase diversity

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17
Q

What are metal cofactors used for?

A

stability or redox

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18
Q

Which metal cofactors are stabilising?

A

High polarity, small size

Mg, Zn

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19
Q

What are the redox metal cofactors?

A

Mn, Fe, Ni, Cu with variable oxidation states

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20
Q

What function do redox metal cofactors have?

A

Act as electron acceptors and can also bind oxygen (Fe)

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21
Q

How are small organic cofactors produced?

A

Derived from vitamins in diet

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22
Q

What are the small organic cofactors?

A 
NAD
FAD
FMN
PLP
CoASH
CoAS-
TPP
Biotin
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23
Q

What enzyme is NAD used in?

A

Alcohol DH

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24
Q

What enzyme is FAD used in?

A

Succinate DH

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25
What is PLP used for?
transamination, racemisation, decarboxylation
26
What is CoASH used for?
Acetyl CoA as leaving group in citrate synthase
27
What is CoAS used for?
Nucleophile in β-oxidation
28
What is TPP used for?
β and α keto acid decarboxylation | pyruvate decarboxylase
29
How does TPP act?
As ylid form to act as an electron sink
30
Which keto acid is more difficult to decarboxylate by TPP?
α as forms a stable anionic intermediate
31
Where is biotin used?
Covalently bound to lysine in pyruvate carboxylase
32
How do enzymes increase reaction rate?
Preferentially stabilise the transition state to lower the activation energy and make -∆G
33
What can transition state analogues be used for?
Competitive inhibitors
34
What does directed evolution do?
Remodels existing enzymes to fit a theoretical TS
35
Are coenzymes permanently bound?
No, they bind and release as substrates
36
What are abzymes?
Catalytic antibodies used in industry to bind TS and lower Ea
37
What are the 4 types of kinetic mechanism?
Ordered sequential bi-bi Random bi-bi Bi-bi ping pong Theorell Chance
38
Which enzyme kinetic mechanisms use ternary complexes?
ordered sequential bi-bi | random bi-bi
39
Example of an enzyme using Ordered sequential bi bi?
NADH-linked dehydrogenases
40
What is an example of an enzyme using bi-bi ping pong?
PLP-dependant transaminases
41
What is an example of Theorell Chance?
Horse liver alcohol dehydrogenase
42
How common is random bi-bi?
Not
43
For ternary complex mechanisms, which kinetic parameter cannot be directly determined?
K'a
44
What additional experiments can be used to determine kinetic mechanism?
Partial reactions Isolation of modified enzyme Equilbrium dialysis Product inhibition
45
Which kinetic mechanism modifies the enzyme?
Bi-bi ping pong
46
What are the rules for product inhibition experiments?
If intercept changes then not acting on E form if slope changes then acting on E form Saturation will make a step irreversible Saturation will make another substrate have a [0]
47
What types of kinetic mechanism can product inhibition be used for?
Ordered sequential bi-bi
48
What type of inhibition changes slope only?
Competitive- only binds E
49
What does non-competitive inhibition change?
Binds E and ES to change slope and intercept
50
What inhibition changes intercept?
uncompetitive
51
What is the structure of Lactate dehydrogenase?
tetameric with different isoforms based on α/β subunit mr of 140kDa 40% α 23%β 2 domains N domain= 6stranded sheet, 4 helices: NADH binding C domain= 2xantiparallel 3 stranded sheet: Substrate binding
52
What kinetic mechanism does LDH have?
ordered sequential of NADH then Lactate
53
Where is the α4 LDH isozyme found?
heart
54
Where is the β4 LDH isozyme found?
Skeletal muscle
55
What is oxamate an analog of?
Pyruvate
56
What is oxalate an analog of?
Lactate
57
What are competitive inhibitors of LDH used for?
X ray crystallography as no turnover
58
How can pre-steady state kinetics be observed?
Continuous flow spectroscopy | Stopped flow is less expensive
59
What does pre-steady state kinetics measure?
The turnover rate (Kcat) in the burst phase of each step to find the rate limiting step
60
What is Kcat?
Vmax/total moles of enzyme
61
What is the rate limiting step of LDH?
Loop closure
62
How can the catalytic mechanism of LDH be observed?
Affinity labels General labels and inactivation Xray crystallography of competitive inhibitors Site directed mutagenesis
63
Which residues are identified in LDH by affinity labels?
His195 is radioactively labelled by nucleophilic attack to bromopyruvate. Unfolded, digested and sequenced
64
What are the general labels used with LDH?
Diethylpyrocarbonate for Histidine Phenylglyoxal for Arginine N-ethylmaleimide for Cysteine
65
How is substrate protection observed in LDH?
NADH and Lactate block inactivation by labelling
66
Which general labels can be sequenced?
Histidine 195 | Cysteine 165
67
How many residues does Phenylglyoxal inactivate?
1Arg per subunit
68
Why is sterochemistry important in catalytic mechanisms?
A/B nicotinamide cofactors | L/D amino acids
69
What is the NADH binding site known as?
The Rossman fold
70
What are the LDH active site residues identified by Xray crystallography?
``` His 195 donates proton Arg 109 excludes water, stabilises developing charge on hydroxyl Arg 171 stabilises carboxyl Lys 250 restricts NAD rotation Asp 168 stabilises Histidine ```
71
What residues are the the LDH loop identified by Xray crystallography?
98-120
72
What has LDH site directed mutagenesis shown?
Loop closure by removing natural fluorescence and adding loop fluorescence which varies dependant on open/closed Loop closure is rate limiting Cys165 is in loop
73
What is the specificity constant?
Kcat/Km
74
What does the trypsin D189K mutation show?
Specificity knockout
75
Which residues are mutated to switch LDH specificity to oxaloacetate?
Overall charge: E107Q exposed, D197N buried Size: T246G: 1800 fold Complementarity: Q102R: 10^7 fold
76
Which residues are mutated to switch LDH specificity to branched substrates?
A235G A236G in α2G JAW | Q102M, K103V, P105S reduce polarity and increase freedom of rotation
77
Do multiple mutations switch specificity with an additive effect?
No- they all all needed
78
Why is Glutathione reductase mutated?
To switch specificity to NADH which is cheaper
79
Which domain mutated in Glutathione reductase to switch specificity?
The Rossman fold
80
Which residues are mutated to switch specificity of Glutathione reductase?
R->M, R->P, K->F, H->D remove phosphate pocket | Second shell: A179G allows A175G to rotate and V->E mutation to enter to stabilise ribose 2'OH
81
How are residues identified for rational protein design?
Using sequence alignments to identify highly conserved residues
82
What switch in specificity is achieved in Glutathione reductase?
18,000 fold

Proteins (12 decks)

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