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Proteins > Enzymes > Flashcards

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
Q

What is PLP used for?

A

transamination, racemisation, decarboxylation

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26
Q

What is CoASH used for?

A

Acetyl CoA as leaving group in citrate synthase

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27
Q

What is CoAS used for?

A

Nucleophile in β-oxidation

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28
Q

What is TPP used for?

A

β and α keto acid decarboxylation

pyruvate decarboxylase

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29
Q

How does TPP act?

A

As ylid form to act as an electron sink

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30
Q

Which keto acid is more difficult to decarboxylate by TPP?

A

α as forms a stable anionic intermediate

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31
Q

Where is biotin used?

A

Covalently bound to lysine in pyruvate carboxylase

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32
Q

How do enzymes increase reaction rate?

A

Preferentially stabilise the transition state to lower the activation energy and make -∆G

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33
Q

What can transition state analogues be used for?

A

Competitive inhibitors

34
Q

What does directed evolution do?

A

Remodels existing enzymes to fit a theoretical TS

35
Q

Are coenzymes permanently bound?

A

No, they bind and release as substrates

36
Q

What are abzymes?

A

Catalytic antibodies used in industry to bind TS and lower Ea

37
Q

What are the 4 types of kinetic mechanism?

A

Ordered sequential bi-bi
Random bi-bi
Bi-bi ping pong
Theorell Chance

38
Q

Which enzyme kinetic mechanisms use ternary complexes?

A

ordered sequential bi-bi

random bi-bi

39
Q

Example of an enzyme using Ordered sequential bi bi?

A

NADH-linked dehydrogenases

40
Q

What is an example of an enzyme using bi-bi ping pong?

A

PLP-dependant transaminases

41
Q

What is an example of Theorell Chance?

A

Horse liver alcohol dehydrogenase

42
Q

How common is random bi-bi?

A

Not

43
Q

For ternary complex mechanisms, which kinetic parameter cannot be directly determined?

A

K’a

44
Q

What additional experiments can be used to determine kinetic mechanism?

A

Partial reactions
Isolation of modified enzyme
Equilbrium dialysis
Product inhibition

45
Q

Which kinetic mechanism modifies the enzyme?

A

Bi-bi ping pong

46
Q

What are the rules for product inhibition experiments?

A

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
Q

What types of kinetic mechanism can product inhibition be used for?

A

Ordered sequential bi-bi

48
Q

What type of inhibition changes slope only?

A

Competitive- only binds E

49
Q

What does non-competitive inhibition change?

A

Binds E and ES to change slope and intercept

50
Q

What inhibition changes intercept?

A

uncompetitive

51
Q

What is the structure of Lactate dehydrogenase?

A

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
Q

What kinetic mechanism does LDH have?

A

ordered sequential of NADH then Lactate

53
Q

Where is the α4 LDH isozyme found?

A

heart

54
Q

Where is the β4 LDH isozyme found?

A

Skeletal muscle

55
Q

What is oxamate an analog of?

A

Pyruvate

56
Q

What is oxalate an analog of?

A

Lactate

57
Q

What are competitive inhibitors of LDH used for?

A

X ray crystallography as no turnover

58
Q

How can pre-steady state kinetics be observed?

A

Continuous flow spectroscopy

Stopped flow is less expensive

59
Q

What does pre-steady state kinetics measure?

A

The turnover rate (Kcat) in the burst phase of each step to find the rate limiting step

60
Q

What is Kcat?

A

Vmax/total moles of enzyme

61
Q

What is the rate limiting step of LDH?

A

Loop closure

62
Q

How can the catalytic mechanism of LDH be observed?

A

Affinity labels
General labels and inactivation
Xray crystallography of competitive inhibitors
Site directed mutagenesis

63
Q

Which residues are identified in LDH by affinity labels?

A

His195 is radioactively labelled by nucleophilic attack to bromopyruvate. Unfolded, digested and sequenced

64
Q

What are the general labels used with LDH?

A

Diethylpyrocarbonate for Histidine
Phenylglyoxal for Arginine
N-ethylmaleimide for Cysteine

65
Q

How is substrate protection observed in LDH?

A

NADH and Lactate block inactivation by labelling

66
Q

Which general labels can be sequenced?

A

Histidine 195

Cysteine 165

67
Q

How many residues does Phenylglyoxal inactivate?

A

1Arg per subunit

68
Q

Why is sterochemistry important in catalytic mechanisms?

A

A/B nicotinamide cofactors

L/D amino acids

69
Q

What is the NADH binding site known as?

A

The Rossman fold

70
Q

What are the LDH active site residues identified by Xray crystallography?

A 
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
Q

What residues are the the LDH loop identified by Xray crystallography?

A

98-120

72
Q

What has LDH site directed mutagenesis shown?

A

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
Q

What is the specificity constant?

A

Kcat/Km

74
Q

What does the trypsin D189K mutation show?

A

Specificity knockout

75
Q

Which residues are mutated to switch LDH specificity to oxaloacetate?

A

Overall charge: E107Q exposed, D197N buried
Size: T246G: 1800 fold
Complementarity: Q102R: 10^7 fold

76
Q

Which residues are mutated to switch LDH specificity to branched substrates?

A

A235G A236G in α2G JAW

Q102M, K103V, P105S reduce polarity and increase freedom of rotation

77
Q

Do multiple mutations switch specificity with an additive effect?

A

No- they all all needed

78
Q

Why is Glutathione reductase mutated?

A

To switch specificity to NADH which is cheaper

79
Q

Which domain mutated in Glutathione reductase to switch specificity?

A

The Rossman fold

80
Q

Which residues are mutated to switch specificity of Glutathione reductase?

A

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
Q

How are residues identified for rational protein design?

A

Using sequence alignments to identify highly conserved residues

82
Q

What switch in specificity is achieved in Glutathione reductase?

A

18,000 fold

Proteins (12 decks)

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