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Y2 Biochemistry > Enzyme Mechanisms > Flashcards

Enzyme Mechanisms Flashcards

1
Q

What are the three things reaction mechanisms determine?

A

Where electrons are
Which bonds are broken/made
Flow of electrons

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2
Q

Describe BrØnsted acids and bases

A

Acid- proton donor
Base- proton acceptor

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3
Q

Describe lewis acids and bases

A

Acid- can form coordinate bonds with lone pairs
Base- has lone pairs available for sharing

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4
Q

What is the pH equation with a certain pKa?

A

pH = pKa + log10([A-]/[HA])

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5
Q

What factors determine acidity of an organic compound?

A

Strength of Y-H bond
Electronegativity of Y
Factors to stabilise Y- compared to YH
Nature of the solvent

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6
Q

What does pKa depend on?

A

Temperature
Ionic strength of solvent
Microenvironment

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7
Q

Describe the protonated and deprotonated forms of histidine

A

The protonated imidazolium cation is stabilised by 2 resonance structures
Deprotonated imidazole has 2 tautomers where either N is protonated

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8
Q

Why is cysteine more acidic than serine?

A

The S valance electrons are further from the nucleus than in O, so electrons are easier to donate

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9
Q

Describe RNase structure

A

V shaped
3-stranded antiparallel β-sheets
N-terminal helix with His12
4 SS bridges

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10
Q

What do RNases cut?

A

They act as endonucleases on ssRNA
Cleave P-O5’ bond
Cuts after a pyrimidine base (U or C), suggesting there must be a recognition site

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11
Q

Describe the key catalytic intermediate of the RNase reaction

A

2’-3’ cyclic nucleotide. This is cleaved in the second step of the RNase reaction

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12
Q

How were kinetic studies used to predict the active site of RNase?

A

The pH profile of Vmax is around 6-7, suggesting there might be 2 His residues in the active site- one acid, one base

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13
Q

How were His12 and His119 identified as active site residues in RNase?

A

Modified RNase could only have one key His modified at a time, but not together, His119 was 88% and His12 12% of the time, so one acted as an acid, the other a base

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14
Q

Describe the RNase A active site residues

A

His12 and His119 are directly involved in cleavage
Lys41 stabilises negatively charged phosphates in the intermediate

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15
Q

Describe the RNase A specificity pocket residues

A

Phe120- van der waals contacts with the RNA base
Ser123 and Thr45 are involved in hydrogen bonding
The pocket is too small for purines (AG)

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16
Q

What is the acidic active site residue of RNase?

A

His12

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17
Q

Give an example of a serine protease

A

Trypsin
Chymotrypsin
Elastase
Thrombin
Kallikrein
Compliment System
Blood clotting factors

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18
Q

What sort of reaction do serine proteases catalyse?

A

Hydrolysis of a scissile bond with water

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19
Q

How do the S1 and S1’ pockets bind to P1 and P1’ respectively?

A

S1 binds upstream and S1’ downstream of the scissile bond

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20
Q

How is chymotrypsin initially synthesized?

A

Inactive enzyme precursor (zymogen) called chymotrypsinogen

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21
Q

Where about in a polypeptide does chymotrypsin cleave?

A

After large aromatic residues (Phe, Tyr, Trp)

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22
Q

What is the catalytic triad in the chymotrypsin active site?

A

Ser195
His57
Asp102

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23
Q

How can Ser195 be modified in chymotrypsin?

A

PMSF blocks serine proteases by modifying Ser195, inhibiting the enzyme

DIPF is a nerve gas which blocks serine proteases and related molecules

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24
Q

How can His57 in Chymotrypsin be identified?

A

TPCK is a substrate analogue which binds at the active site and is nucleophilic attacked by His

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25
Q

Describe trypsin-like serine protease structure

A

Two β-barrels formed from 6 anti-parallel β-strands
His57, Ser195, Asp102 catalytic triad
Oxyanion hole
Specificity pocket
Main chain substrate binding

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26
Q

Describe how the chymotrypsin catalytic triad is stabilised

A

-ve Asp102 stabilises formation of +ve His57, so the proton from Ser195 can donate to His57
This makes Ser195 nucleophilic

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27
Q

Describe the chymotypsin oxyanion hole

A

Located near the carbonyl of the substrate scissile bond
Backbone amide hydrogens of Ser195 and Gly193 point to the active site to stabilize the tetrahedral enzyme-substrate intermediate

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28
Q

Describe the chymotrypsin mechanism

A
  1. His57 attacks Ser195 which attacks the carbonyl group of the substrate, forming a covalent bond. This forms a -ve tetrahedral intermediate stabilised by the oxyanion hole
  2. Tetrahedral intermediate decomposes to the acyl-enzyme intermediate with His57 donating the proton
  3. The enzyme is deacylated through another tetrahedral intermediate. Water is the nucleophile, Ser195 is the leaving group
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29
Q

What % identity do chymotrypsin and trypsin have?

A

40%

30
Q

What residues are found in the chymotrypsin, trypsin and elastase specificity pockets?

A

Chymotrypsin: G266, S189, G216
Trypsin: G266, D189, G216
Elastase: V226, T216

31
Q

How do serine proteases orient polypeptides for cleavage?

A

Bind to the side chains in the specificity pocket and recognise the main chain conformation.

32
Q

How are serine proteases controlled?

A

Produced as zymogens (proenzymes) which require proteolytic cleavage of their precursors to activate

33
Q

What is the protease activation cascade master activator?

A

Eteropetidase

34
Q

how is α-chymotrypsin activated?

A
  1. Chymotrypsinogen is partially activated by cleavage by trypsin to π-chymotrypsin.
  2. This activates other π-chymotrypsin molecules to form α-chymotrypsin.
  3. The A,B and C chain are connected by disulfide bonds
35
Q

How is the chymotrypsin active site activated?

A

The N-terminal Ile16 on π-chymotrypsin ionically pairs to the Asp194 side chain.
This changes the position of the Ser195 side chain for the correct geometry.
The amides of residues 193 and 195 are oriented to form the oxyanion hole

36
Q

Where does thrombin cleave fibrinogen?

A

Arg-Gly
This forms plaques of insoluble fibrin, forming clots

37
Q

How are serine proteases used in the blood clotting cascade?

A

Thrombin cleaves fibrinogen to form insoluble fibrin
Positive and negative feedback form the activation and deactivation systems
Plasmin/plasminogen system dissolves clots

38
Q

Give an example of convergent evolution of serine proteases

A

Subtilisin is a bacterial protease with an unrelated sequence to chymotrypsin. However it has the Asp, His, Ser catalytic triad

39
Q

Describe proteasomes

A

Intracellular complexes with proteolytically active sites in its interior cave belonging to N-terminal threonine hydrolases.
4 stacked rings of 7-fold symmetry

40
Q

What sort of activity do the protease active sites of the inner (β) subunits of the proteasome have?

A

β1- caspase
β2- tryptic
β5- chymotryptic

41
Q

How does bortezomic inhibit apoptosis?

A

Induces the ubiquitin-proteasome pathway by binding to the β5 subunit.
It has a trigonal conformation which mimics a peptide bond.
When boron is attacked by a nucleophile, it is converted from trigonal sp2 to tetrahedral sp3.

42
Q

What cellular processes are associated with cysteine proteases?

A

Apoptosis, parasitic infections, virus maturation

43
Q

Outline the action of a cysteine protease

A
  1. Nucleophilic attack by a thiolate on C=O
  2. Tetrahedral intermediate forms in the oxyanion hole
  3. A water molecule is activated by His and attacks the carbonyl group
  4. Second tetrahedral intermediate forms
44
Q

What is Burkoldria?

A

B. pseudomallei. Potential bioterror weapon.
Multi-drug resistant bacterium with no vaccine.
Infection mimics TB
7 morphotypes with different patterns of gene expression

45
Q

How does BPSL1549 from burkholdria compare to the CNF1 domain?

A

They have structural, but not sequence similarity. This is an example of convergent evolution.
The catalytic His-Cys pair is conserved

46
Q

Describe CNF1

A

114kDa pathogenic toxin expressed by some E.coli
Deamidates key glutamine residues in small GTPases (Rho, Rac and Cdc42)
Inhibits GTP hydrolysis, by deactivating GTPases. This leads to cytoskeleton remodelling

47
Q

Describe BPSL1549

A

23kDa protein in Burkholdria.
Unknown function and no sequence similarity to proteins outside of burkholderia.
Deamidates Gln339 in RNA helicase eIF4A, preventing initiation-dependant protein translation.

48
Q

What metal ion facilitates catalysis of the enolase superfamily?

A

Mg2+

49
Q

Outline the mandelate racemase mechanism

A

Lys166 acts as a base to remove H from the chiral carbon.
The OH then attachs the carbonyl C=O
His297 acts as an acid and returns the proton

50
Q

Describe the mandelate racemase active site

A

The Mg ion is coordinated by carboxylic acid groups on Asp/Glu
Mg2+ stabilises the acid-carboxylate
The L-proton faces Lys166 and catalytic histidine returns the D-proton on the opposite side

51
Q

What is a common H- source in lab settings

A

BH4- (Borohydride)

52
Q

Why are biological reductants better than BH4-?

A

They use cofactors which are directed by enzymes to target molecules, making them very specific

53
Q

Describe the main features of an NAD ring

A

Net positive charge all over
Phosphate backbone in nucleotides gives an overall negative charge

54
Q

Why is hydride transfer very stereospecific?

A

The enzyme differentiates between hydrogens pro-R/pro-S on the carbon.
Hydride transfers to a particular face of the nicotinamide ring

55
Q

What parameters are critical to hydride transfer?

A

Distance and angle of approach of hydride.
In NADH, this is 3-3.5Å, also known as van der Waals distance

56
Q

What are the basic steps in fatty acid biosynthesis?

A

Oxidation -> hydration -> oxidation

57
Q

Describe type I fatty acid synthase

A

Contain catalytic domains 1 and 2
Found in bacteria, vertebrates and yeast

58
Q

Describe type II fatty acid synthase

A

Contain discrete polypeptides catalysing each enzymatic step
Found in plants and many bacteria

59
Q

How do acyl carrier proteins bring acyl groups to fatty acid synthase?

A

Its phophoantetheine covalently linked to a serine residue ( S-acyl primed) attacks the acyl group.

60
Q

What is the first step of fatty acid elongation?

A

Oxidation: uses β-keto ACP reductase
NADP+ reduced
tetrameric to trigonal planar

61
Q

Describe the β-keto ACP reductase (BKR) active site?

A

Rossman fold and S…YxxxK motif.
Tyr and Lys in the active site

62
Q

Describe Bassica napus ENR (enoyl-acyl carrier protein reductase)

A

Isoform from developing rapeseed seeds
Isolated from chloroplast
Tetramer with each subunit being 32.5kDa
NADH specific

63
Q

How do the structures of BKR and ENR compare?

A

High structural similarity despite <20% sequence identity
Active site tyrosines do not directly superimpose
Phenyl hydroxyl groups are very close in position

64
Q

Outline the ENR mechanism

A

Hydride transfer from NADH to C3 at a double bond in the acyl substrate
Rearrangement forms an enolate anion
A proton is donated from tyrosine side chain
Tautomerisation from Enol to keto to give a reduced product

65
Q

How is the ENR active site postitioned?

A

Positioned for the nicotinamide ring to go in

66
Q

What does a G93 to V93 change in enolate reductases (ENR) cause?

A

Not much change as van der waals forces overlap

67
Q

How can phosphodiester bond hydrolysis be described?

A

SN2:
Substitution
Nucleophilic
2 molecules (bimolecular)

68
Q

What is the associative mechanism of phosphodiester bond cleavage?

A

Water attacks phosphate
HOR group leaves
Trigonal pyramidal intermediate

69
Q

What is the dissociative mechanism of phosphodiester bond cleavage?

A

Water molecule sits close to the phosphate
Trigonal planar intermediate forms
OR group leaves

70
Q

How can RNA phosphodiester bonds self-cleave?

A

Alkaline conditions
Base attacks 2’ OH on ribose which attacks 3’ end of phosphate

71
Q

What are the key parts of RNA nucleases?

A

His12 cleaves phosphate group
His119 accepts the proton

Y2 Biochemistry (7 decks)

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