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Protein structure and function > Enzymes > Flashcards

Enzymes Flashcards

1
Q

What does TIM stand for?

A

Triose Phosphate Isomerase

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2
Q

what is the role of TIM

A

it catalyses the interconversion of dihydroxyacetone and glyceraldehyde 3 phosphate through an enzyme bound enediol intermediate.

G3P to DHAP in glycolysis

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3
Q

What process is TIM centeral to?

A

Glycolysis.

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4
Q

WHat is the struture of TIM?

A

(BetaAlpha)8 - barrel (8 subunits of betaAlpha)

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5
Q

What is the construction principle of TIM?

A

beta strand followed by an alpha helix and this motif is repeated 8 times.

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6
Q

Where is the active site located in TIM?

A

at the end of the beta strands. beta strands are parallel to each other at the active site.

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7
Q

How is TIM involved in Glycolysis?

A

It is needed to convert DHAP to G3P. There is no regulation it just needs to be as quick as possible

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8
Q

What occurs in the conversion of DHAP to G3P?

A

Isomerisation reaction is catalysed where protons are moved from C1 to C2 via internal reDOx with oxidation of C1 and reduction of C2.

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9
Q

describe the steps in the REDOX of DHAP to G3P

A
  1. base 1 (B1-) abstracts the proton at C1.
  2. Donation of proton by acid A1 at C2=O
  3. Donation of proton by acid A2 at C2.
  4. abstraction of proton by base B2- at C1OH
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10
Q

between G3P and DHAP where does the free energy equlibrium stand?

A

free energy is higher for Enzyme+G3P but DHAP still gets converted as even though the equlibrium stands with DHAP because G3P keeps getting removed for glycolysis it still gets made.

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11
Q

How many intermediates are there between DHAP and G3P?

A

3 intermediates.

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12
Q

what is the equalibrium ratio between DHAP and G3P

A

96:4 for DHAP:G3P

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13
Q

What are some amino acids used in acid base catalysis?

A 
Glu, Asp
Lys, Arg
Cys
His
Ser
Tyr
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14
Q

What amino acids in TIM act as the base and Acid? what are the steps inolved in the movement of the protons

A

Glu165 acts as B1- (glutamic acid)
His95 acts as Acid A1H (histidine)

when glu165 takes the H+ it becomes acid2 (A2H)
His95 is restored to A1H.

Glu165 is restored to B1- when it donates the H+ to C2.

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15
Q

what is the difference between convergent and divergent evolution?

A

Convergent shows different functions converge into the same fold.

divergent shows common ancestors structure diverge into different functions.

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16
Q

What is the case that was hypothesised for gene duplication?

A

Gene duplication generates 2 identical half barrels that fuse to become a (ba)8 barrel. gene duplication diversifies it into 2 enzymes with distinict catalytic activities. (HisA and HisF)

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17
Q

What are some of the physiological reactions mediated by proteases?

A

digestion of food

regulation of cascades e.g blood clotting or apoptosis

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18
Q

name two serine protease triad enzymes and how their strucutures differ?

A

Trypsin and Subtilisin.

There is no sequence or structual homology between triad enzymes.

therefore triad enzymes are a strong case for convergent evolution.

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19
Q

What amino acids make up a triad enzyme.

A

Tend to be serine, histidine and aspartate

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20
Q

what is an oxyanion hole?

A

Oxyanion holes are commonly found in many enzyme structures. They are crucial for the stabilization of high-energy oxyanion intermediates or transition states through hydrogen bonding.

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21
Q

in Subtilisin triad enzyme what amino acid is important for the oreientation of the enzyme? how was this discovered?

A

Aspartate. was discovered by mutagensisis studies that got rid of one of the amino acids to see if it still worked without it.

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22
Q

What does the Oxyanion hole do?

A

Lowers teh pKa of the oxyanion which stabalises the transition state.

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23
Q

What are the components needed in a triad protease

A

Catalytic triad
substrate peptide binding cleft
make oxyanion hole
specicifity pocket

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24
Q

What is subtilisin?

A

a serine protease that has a high stability with low substrate specificity.
has simplified purification and secreted by the Bacillius.

has a molecular weight of 17-90kDa and is found in most laundry and dishwasher formulations.

degrade stains made of proteins such as milk, egg, grass, sauces, blood.

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25
Q

All signal peptidases are serine proteases. how do they work?

A

cleave N terminal signal peptides. can be:

ER targeting
Bacterial leader-peptidases
Mitrocondrial signal peptidases of the inner mitocondrial membrane

they move signals across membranes and regulate transport and trafficking of protein in the cell.

26
Q

describe the blood clotting cascade

A

coagulation cascade results in the acumulation of platlets at the wound site and the formation of a fibrin clot.
uses serine protease Thrombin and factors VII, IX, X, XII (7,9,10,11)

Plasmin is the key enzyme in the fibrinolytic cascade for disolving the blood clots by acting as a nonspecific proteease to trigger a variety of proteins in the plasma and tissues aswell as the blood clot destruction

27
Q

What is a cystine protease?

A

they have a catalytic dyad with a cystine as a neucleophile. examples of them are

papain
bromelain
caspase.

they are stronger nucleophile than serine so only need 2 residues

28
Q

What is a caspase

A

is a cystine protease that leaves proteins at the carboxy-terminal side of asp residues. (C- Asp)

involved in controlled cell death/Apoptosis. (proteases of death!)

also involved in differentiation, proliferation, immune system regulation

a neurogenative disease involving caspases is chorea Huntington.

29
Q

What are the 3 intermediates formed by TIM?

A

E.DHAP (enzyme substrate)
E.enediol (enzyme and intermediate)
E.G3P (enzyme product)

(E is enzyme)

30
Q

what is the transition state theory?

A

enzymes stabalise the transition state and lower the activation energy. in a catalysed reaction the rate changes, not the equilibirum.

31
Q

how are proteolytic cascasdes activated?

A

by clevage of the peptide

32
Q

what is Ubiqutin?

A

a small 76 amino acid regulatory protein

33
Q

where is ubiqutin found and what is its function?

A

found in all eukaryotic cells and directs the movment of important proteins in the cell. participates in both synthesis of new proteins and the destruction of defective proteins.

34
Q

what is the triad protease enzyme cap made of what what do they do?

A

made up of two proteins.

Rpn: senses ubiqutination state
Rpt: ATPase uses to close the lid of

35
Q

why are serine triad proteases a good case for convergent evolution?

A

Trypsin and subtilisin are both serine proteases. there is no sequence or structual homology between the triad enzymes. indicates convergent evolution

36
Q

what is the serine triad subtilsin active site mconfigured with?

A

serine, histidine, aspartate

37
Q

what did the mutatgenisis studies determine about the acid in the triad?

A

needed for the orientation of the triad.

38
Q

what is the oxyanion hole?

A

a hole to stabalise the transition state. acts by lowering the pKa of the oxyanion

39
Q

what is a triad made of?

A

nucleophile, base and acid

40
Q

what is the rate limiting step in the triad mechanisim?

A

the formation of the first tetrahedral intermediate

41
Q

in what common household use is the serine protease subtilisin found?

A

in most laudry and dishwasher formulations. they can degrade proteinaceous stains (milk, egg, grass, sauces, blood)

42
Q

what type of proteases are signal peptidases?

A

serine proteases

43
Q

what causes the coagulation cascade (blood clotting cascade)

A

accumulation of platlets at the wound site and the formation of a fibrin clot that stems blood flow in order to limit blood loss.

44
Q

what is the serine protease in the blood clotting cascade and its factors?

A

Thrombin and factors VII, IX, X, and XI

45
Q

what is plasmin?

A

the key enzyme in the fibrinolytic cascade disolving blood clots. functions as a non specific protease that triggers degradeation of a variety of proteins in plasma and tissues as well as destroying blood clots.

46
Q

why are cystine proteases dyads not triads?

A

proton is lost much more easily in cystine than serine so 3rd residue acid is not needed. its a stronger nucleophile.

47
Q

what are some examples of cystine proteases?

A

papain, bromelain, caspase.

48
Q

the proteasome is made up of lots of subunits. what subunits are active in their enzymatic activity?

A

beta 1 and 2 and 5. the subunits are triad enzymes which have threonine as the nucleophile.

49
Q

what is the difference in the peptidoglycan layer of Gram positive and Gram negative bacteria?

A

Gram positive have much thicker multi-layer peptidoglycan layer. (cell wall)

50
Q

what enzyme is needed to synthesie peptidoglycans

A

transpeptidase

51
Q

how does transpeptidase work?

A

has a serine residue with an OH nucleophile, forms a covalent inermediate with the chain.
allows the glycan chain to bind to the first chain at the C=O bond of the amino acid the serine bound to.

52
Q

how does penicillin affect transpeptidase to work as an antibiotic?

A

the beta-lactam of the penicilin forms a covalent enzyme adduct (direct sum of two molecules)

this irrevesibly inactivates the transpeptidase that synthesises the peptidoglycan

53
Q

how do beta-lactamases lead to antibiotic resistance?

A

cleaves the beta lactam which inactivates the penicillin which can lead to antibiotic resistance.

54
Q

what sort of enzyme is beta lactamase?

A

a triad enzyme.

55
Q

what is a second line antibiotic that targets beta lactamase?

A

Clavulanic acid. forms a covalent adduct that irrevesibilly inactivates the enzyme beta lactamase so penicillin cannot be inactivated.

56
Q

what are matrix metalloproteases involved in? (MMP)

A

signalling, morphogensis, and wound healing

57
Q

what domains are the alzheimer Precursor protein made of?

A 
SP-signal peptide
HB-heparin binding domain
CB-copper binding domain
A-acidic domain
E2
Abeta-amyloid-beta domain
ACID-APP intracellular domain
58
Q

where is the APP (alzhimers precurosr protein) found?

A

in the synpanses.

59
Q

what is the non-amylodogenic pathway?

A

the alpha secretase which is a metalloprotease cuts the protein. then gamma secretase that is an aspartate protease cuts the p3 segment off the protein.

60
Q

what is the alzheimers sympotmatic pathway?

A

first beta secretase cuts the protein. is a serine protease. then gamma secretase cuts which leaves teh Abeta segment that produces amalyoid forming the senile plaque which is in alzhiemers.

61
Q

why does trypsin only cleave at the carboxyl end of peptides?

A

the s1 substrate binding pocket as an aspartate negative residue which means it will be specific for the positive resuides such as Arg and Lys.

Protein structure and function (7 decks)

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