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Biochemistry 2 > Rice > Flashcards

Rice Flashcards

1
Q

What are isozymes?

A
  • Enzymes that have the same function but have different amino acid sequences
  • They can often be distinguished by differences in optimal pH, kinetic properties or immunology
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2
Q

How are enzymes classified/names?

A
  • Often name is derived from the substrate/chemical reaction that it catalyses
  • EC numbers are used as nomenclature, each enzyme is described by a sequence of four numbers headed by EC
  • The first number broadly classifies the enzyme based on its mechanism
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3
Q

What is an alkoxide?

A

An alkoxide is the conjugate base of an alcohol and therefore consists of an organic group bonded to a negatively charged oxygen atom

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4
Q

What happens if an alcohol group loses its proton?

A

It becomes an alkoxide

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5
Q

How can we stabilise negative charges for instance an alkoxide ion?

A
  • We can place them in an environment whereby there is a positive charge adjacent to the negative charge
  • This encourages the loss of the proton
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6
Q

What type of catalysis do serine proteases display?

A

Nucleophilic covalent catalysis

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7
Q

What is the overall function of serine proteases?

A

To cleave polypeptides (hence protease)

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8
Q

What were the four evolutionary ideas about enzyme catalysis?

A
  1. Fischer - Lock and Key theory
  2. Haldane - Stickase enzyme which supposedly breaks sticks in two (all hypothetical) - this involved a transition state of a bent stick
  3. Linus Pauling - enzymes as molecules that are complementary to the activated complex of the reactions that they catalyse
    - Enzyme would form strong bonds to the activated complex but only weak bonds with the products so these could diffuse off
  4. Koshland - Induced fit model; active site was not preformed and only formed when the substrate was bound
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9
Q

What makes a reaction more irreversible?

A

The larger the net free energy change

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10
Q

What makes a reaction slower?

A

The larger the activation energy

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11
Q

What is the reaction coordinate?

A

Term used to describe the progress of a reaction from starting materials to products

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12
Q

What are the key characteristics of an exothermic reaction?

A

An exothermic reaction has products of lower free energy than reactants

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13
Q

Why can the transition state in a reaction not be isolated?

A

It is a transient structure which represents the peak of the energy profile as the reaction proceeds

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14
Q

What is the activation energy?

A

The difference between the energy of a transition state and reactants

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15
Q

How does an energy diagram for a 2 step reaction differ from that of a one step reaction?

A

An intermediate is formed in the conversion of reactants to products

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16
Q

What was Pauling’s idea about enzyme catalysis?

A

Enzyme catalysis results from lowering the energy of the transition state by more than the lowering of the energy of reactant or products

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17
Q

What sort of catalysis do enzymes use?

A
  • Covalent catalysis (nucleophilic and electrophilic)
  • Acid catalysis
  • Base catalysis
  • Metal ion catalysis
  • Strain catalysis
  • Conformational organisational catalysis (entrophic control)
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18
Q

What is the functional group of cysteine?

A
  • CH2-SH
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19
Q

What enzymes is cysteine associated with?

A

Thiol proteases and GAPDH

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20
Q

What is a summary of the chymotrypsin mechanism?

A

Chymotrypsin uses the CH2-OH group of SER-195 (key residue) as a nucleophilic covalent catalyst to form an acyl-enzyme intermediate and uses His-57 as a proton donor/ acceptor in the reaction

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21
Q

Why is the mechanism of chymotrypsin a nucleophilic covalent catalysis reaction?

A

As in the active site there is a serine which is a nucleophile in the chemical process

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22
Q

What is the role of a protease?

A

Hydrolyse peptide bonds to get an additional C terminus and an amino group

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23
Q

What is a scissile bond?

A
  • Covalent chemical bond that can be broken by an enzyme

- In a peptide it is the C-N bond of the amine

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24
Q

How does subtilisin differ from trypsin?

A
  • It also has a serine residue in its active site, however it differs from trypsin as the 3D structure is completely different
  • Good example of convergent evolution
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25
Q

What are the key functional groups for metaloid proteases?

A
  • Zinc ion and aspartate residue (Zn2+, Asp-COOH)

- Enzyme examples: Carboxypeptidase A

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26
Q

What are aspartate protease active site residues?

A
  • Aspartates…DUH (Asp H and Asp -)

- Enzyme examples: Pepsin, chymosin

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27
Q

What are the key active site residues for thiol or cysteine proteases?

A
  • Cysteine residues (Cys-SH)

- Enzyme examples: Papain and actinidin

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28
Q

What are the two main categories of serine proteases?

A
  • Trypsin like (Ser-OH)

- Subtilisin like (Ser-OH)

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29
Q

What are some examples of serine proteases?

A
  • Trypsin
  • Chymotrypsin
  • Elastase
  • Thrombin
  • Plasmin
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30
Q

What are the key active site residues in glutamate peptidases?

A
  • Glu-COOH

- Enzyme examples: Fungal endopeptidases (eqolysins)

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31
Q

What are the key active site residues in threonine proteases?

A
  • Thr-OH

- Enzyme examples: Proteasome

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32
Q

What are the main general roles of proteases?

A

They have main functions in digestion/ blood clotting and therefore are targets for drug treatments

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33
Q

What do substrate specificity pockets do?

A

Select for certain types of side chain

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34
Q

What pockets are upstream of the scissile bond in proteases?

A

Pockets S1, S2 and S3

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35
Q

What pockets are downstream of the scissile bond in proteases?

A

Pockets S1’, S2’ and S3’

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36
Q

What does pocket S1 bind?

A
  • Pocket S1 binds residue P1-P1 which is the main specificity site after which cleavage occurs
  • P2 fits into S2 and so on
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37
Q

What is the main specificity site in proteases?

A

S1

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38
Q

What is the specificity group after which cleavage occurs in trypsin?

A

Arg and Lys

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39
Q

What is the specificity group after which cleavage occurs in chymotrypsin?

A

After an aromatic or hydrophobic residue e.g. Phe, Tyr and Trp

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40
Q

What is the specificity group after which cleavage occurs in Elastase?

A

After a small residue such as alanine

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41
Q

What is the specificity group after which cleavage occurs in Thrombin?

A

Arg and Gly

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42
Q

Why would presence of aspartate not work in the chymotrypsin active site?

A

You are putting a negative charge next to lots of hydrophobic residues therefore it cannot be deprotonated

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43
Q

Can Alanine fit into the specificity pocket of chymotrypsin?

A

Yes it can but not very well

  • The affinity is low and we can improve this by making the specificity pocket smaller
  • Residues in the specificity pocket are modified to things like valine to bulk out the specificity pocket
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44
Q

What is the key essential residue in the chymotrypsin mechanism?

A

Ser 195 and His 57

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45
Q

What is PMSF and what is its function?

A
  • Phenyl methane sulfonyl chloride
  • It is used in preps to block serine proteases
  • It modifies the essential residue in Chymotrypsin (Ser 195) totally inhibiting the enzyme
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46
Q

What is DIPF and what is its function?

A
  • Di isopropyl phospho fluoridate
  • Blocks serine proteases and related molecules, e.g. acetyl cholinesterase involved in synaptic transmission in CNS
  • Nerve gas
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47
Q

What is the mechanism of DIPF in the active site?

A
  • The compound has a fluorine that is very chemically reactive
  • Therefore it can be displaced with the serine in the active site
  • The fluorine leaves and a covalent complex is formed (sulfur attaches to the oxygen)
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48
Q

How do you make a nucleophile?

A

You remove the proton to make an O- group, e.g. in serine protease the Ser195 residue can be deprotonated in order for it to become a nucleophile

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49
Q

What does the negative Asp 102 in the Serine protease catalytic triad do?

A
  • It stabilises the adjacent positive His 57 residue which helps the His 57 grab an extra proton from the Ser 195
  • This makes Ser 195 nucleophilic and so it is highly reactive against substrates or inhibitors
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50
Q

Without Asp 102, what is the rate of catalysis?

A

Without Asp 102, the rate of catalysis is only 0.05% of WT

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51
Q

Where is the oxyanion hole located near?

A

The carbonyl group of the substrates scissile bond

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52
Q

What does the oxyanion hole name denote?

A

A region in the active site where the backbone amide hydrogens of Ser195 and Gly193 point into the active site cavity

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53
Q

How much does the oxyanion hole increase the enzyme activity factor?

A

By 10,000 times

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54
Q

How much sequence identity does chymotrypsin (5CHA) and Trypsin (5PTP) share?

A

40% sequence identity

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55
Q

What is 5CHA?

A

The code for the structure of chymotrypsin

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56
Q

On a sequence identity figure, what do the stars indicate?

A

Where the residues are identical

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57
Q

On a sequence identify figure, what do the dots indicate?

A

Conservative substitutions

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58
Q

What is a conservative substitution?

A

What is a conservative substitution?
An amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size), e.g. Leucine to isoleucine

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59
Q

What happens if you increase the sequence identity?

A

The folds get more and more similar

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60
Q

Where are insertion and deletion mutations usually found in enzymes?

A

In the loops that join the regions of regular secondary structure together

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61
Q

What kind of side chain does trypsin recognise in its specificity pocket and what does this determine?

A

Trypsin recognises a positively charged side chain (lysine) in its specificity pocket, this means that it probably has a negatively charged residue making up the specificity pocket which recognises the amino group of lysine

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62
Q

What is the result of glycine bulking out of the specificity pocket in serine protease?

A
  • Side chain of glycine is just a hydrogen so it is fairly small
  • This creates space so that methyl groups can be in the pocket
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63
Q

Is chymotrypsin hydrophobic or hydrophilic?

A

Hydrophobic

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64
Q

What residue does chymotrypsin have instead of a glycine?

A

An alanine

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65
Q

Why do you remove the negatively charge from Ser in chymotrypsin?

A

So that the specificity pocket can bind hydrophobic groups

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66
Q

Where other than the side chain does the enzyme have to bind to on the substrate?

A
  • The substrate backbone (Enzyme makes interactions with the whole thing, NOT just the small section that does all the chemistry)
  • This helps orientate the substrate for cleavage
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67
Q

How are more specific serine proteases different from normal serine proteases?

A
  • They have more elaborate side chains and main chain recognition sites
  • This means that they can only recognise very specific residues in their specificity pockets compared to other serine proteases
  • This limits the number of substrates they can bind to, e.g. some may only be able to cleave after a lysine which is specifically followed by a tryptophan
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68
Q

Why must enzymes such as chymotrypsin be closely controlled?

A
  • Enzymes such as chymotrypsin destroy other proteins and therefore must be closely controlled
  • Otherwise digestive enzymes would digest your own intestine
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69
Q

What are enzymes such as chymotrypsin first produced as?

A
  • Inactive zymogens or proenzymes

- They need proteolytic cleavage of their precursors to activate them

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70
Q

Why does proteolytic activation require close control?

A

As it is not reversible, so you wouldn’t want to accidentally activate something that could do lots of damage, there is therefore use of inhibitor proteins

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71
Q

How is proteolytic activation controlled in trypsin?

A

Control is provided by pancreatic trypsin inhibitor which forms a very tight interaction with the enzyme, thus inhibiting its activity

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72
Q

How is trypsin first produced?

A
  • Trypsin is first produced as trypsinogen which is cleaved by another enzyme called enteropeptidase
  • The proteolytic fragments are produced in the active trypsin
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73
Q

What can trypsin do to proelastase?

A

Turn it into elastase

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74
Q

What is the proenzyme of chymotrypsin?

A

Chymotrypsinogen

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75
Q

What is subtilisin?

A

A bacterial protease

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76
Q

What is specific acid-base catalysis?

A

Proton is fully transferred before the covalent bonds make/break

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77
Q

What is general acid-base catalysis?

A
  • Where proton is transferred at the same time as covalent bonds are being made
  • This is what enzymes mostly use
  • Amino acid that acts as an acid in the forward reaction acts as a base in the reverse reaction
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78
Q

What are the typical amino acid side chains used in general acid base catalysis?

A

Histidine, Aspartate, Glutamate, Tyrosine, Lysine and Cysteine (v much less common)

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79
Q

What is General acid base catalysis an example of?

A

Microscopic reversibility

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80
Q

What are proteasomes?

A
  • Intracellular multisubunit, cylinder shaped complexes with an interior cave containing proteolytically active sites
  • These proteolytic active sites belong to the N terminal threonine hydrolase
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81
Q

How many subunits are there in a proteasome?

A

14 alpha subunits and 14 beta subunits

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82
Q

Which of the subunits have proteolytic activity in the proteasomes?

A

The beta subunits

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83
Q

What are two types of proteasomes you can get?

A

20S and 26S

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84
Q

What is at the heart of the proteasome?

A

Core particle of 28 protein subunits arranged in 4 stacked rings with 7-fold symmetry

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85
Q

In the proteasome, what beta subunits have catalytic activity and what kinds of catalytic activity do they have?

A

Beta 1, beta 2 and beta 5 have caspase, tryptic and chymotryptic like activity

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86
Q

What type of catalytic activity cleaves after acidic residues?

A

Caspases

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87
Q

What type of catalytic activity cleaves after basic residues?

A

Trypsin

88
Q

What type of catalytic activity cleaves after hydrophobic residues?

A

Chymotryptic like

89
Q

How is threonine similar to/ different from Serine proteases?

A

It is like serine however histidine isn’t the only base, you can also have LYSINE

90
Q

What peptidase is at the heart of the proteasome?

A

A threonine peptidase

91
Q

How does a threonine peptidase work?

A

Threonine at the N term of the Beta subunit acts as the attacking nucleophile assisted by the sidechain of a neighbouring lysine residue (and the N terminal amino group)

92
Q

What base is used to pull a proton off the threonine?

A

A lysine is used as NH2 gives a lone pair that is the base which can pull off a proton which activates the nucleophilic serine

93
Q

Where is the acyl enzyme intermediate in the reaction?

A

The acyl enzyme intermediate is with the threonine side chain rather than the serine

94
Q

How is the acyl enzyme intermediate solved?

A

With a water molecule

95
Q

What inhibitor targets the proteasome?

A

Bortezomib

96
Q

What does the ubiquitin proteasome pathway play a role in?

A

Cytoplasmic turnover

97
Q

What were some proteasome inhibitors found to exhibit?

A

some apoptosis in tumour derived cell lines

98
Q

What is bortezomib?

A

Dipeptide boronic acid

99
Q

What types of cancers is bortezomib used to treat?

A

Multiple myeloma and other cancers

100
Q

What is the chemistry of boron?

A
  • Boron is an electron deficient atom
  • Its electron shell can be completed by attack of a nucleophile forming a covalent bond and converting the Boron from a flat trigonal sp2 hybridisaton to sp3 tetrahedral geometry
101
Q

How do boron inhibitors of enzymes often work?

A

React with an active site nucleophile creating a negative charged boron atom covalently linked to an enzyme

102
Q

Where does bortezomib bind in the proteasome?

A

The catalytic beta subunits

103
Q

What is bovine CPA?

A

Zinc exopeptidase

104
Q

What does bovine CPA catalyse?

A
  • Catalyses the hydrolysis of C-term amino acids from polypeptide chains, showing a preference for susbstrates with aromatic side-chains, e.g. particularly if the residue in front is a Phe
  • This releases an amino acid
105
Q

What does a metal ion encourage?

A

The stabilisation of negative charge (just like the oxyanion hole does for serine proteases)

106
Q

How is the zinc ion coordinated in the active site?

A

Tetrahedrally coordinated to 3 protein residues: 2 histidines and a glutamate AND the 4th ligand position is taken by water

The oxygens and the nitrogens are good ligands to the zinc

107
Q

Does Zinc prefer to bind to OH- or H2O?

What does this say about the pKa of the water molecule?

A
  • Zinc prefers to bind to OH- rather than H2O
  • This means that the pKa of the water molecule is going to change as a result and that the water molecule would LIKE TO BE AN OH-
108
Q

What is the OH- role (that has been generated from the water)?

A

The OH- is then a powerful nucleophile

109
Q

What residue helps the water molecule to become an OH-?

A

Glutamate 270 (acts as an attacking nucleophile) is next to the water molecule and removes the proton to give OH-

110
Q

Where does the OH- attack?

A
  • The key centre in the peptide chain

- Does it in one step rather than two

111
Q

What is the nucleophile in the glutamate endopeptidase reaction?

A

The water molecule hydrogen bonded to the Glu136 and Gln53

112
Q

What is the general base in the glutamate endopeptidase reaction?

A

Carboxylate of Glu136

113
Q

What stabilises the tetrahedral intermediate?

A

The side chain of Gln53 assists in the nucleophilic attack and stabilises the tetrahedral intermediate by HYDROGEN BONDING

114
Q

What could glutamate endopeptidases potentially be used for?

A

Antifungals

115
Q

What is the nucleophile in cysteine proteases?

A

A side chain of the cysteine residue (NOT ser or thr)

116
Q

What is the pKa of the cysteine residue?

A

The pKa is 8 so it is relatively easy to pull the proton off compared to something like ser which has a pKa of 15/16)

117
Q

What is a type of cysteine protease?

A

Papain

118
Q

What does papain use?

A

The -SH group of cysteine and imizadol ring of His 159 to form an ion pair

119
Q

In cysteine proteases, what attacks the peptide bond that wants to be cleaved?

A

The nucleophilic thiolate anion attacks the peptide bond

120
Q

What is formed following the release of the first product?

A

The acyl enzyme intermediate

121
Q

What is used in the second half of the reaction to form the second product?

A

Water is used as a nucleophile to hydrolyse the thiol ester and form the carboxylate as the second product

122
Q

What is a lipase?

A

Lipase is an esterase capable of cleaving the fatty acid chain from glycerol backbone of a triglyceride

123
Q

What kind of reaction is lipase cleavage?

A

A hydrolysis reaction

124
Q

What is the lipase enzyme a target for?

A

Controlling obesity

125
Q

What do pancreatic lipases hydrolyse?

A

Triacylglycerol to 2-acylglycerol

126
Q

What is the structure of a triglyceride?

A

Glycerol backbone with 3 hydroxyls, each of which are linked to fatty acids by an ester bond

127
Q

What is the chemistry of a lipase similar to?

A

An esterase as the oxygen is a good leaving group

128
Q

What is the overall reaction of the lipase?

A

Essentially the lipase will remove the fatty acid group to give the diacylglycerol and so on to give the monoglycerol

Triacylglycerol –> Diacylglycerol –> Monoacylglycerol

129
Q

What similarities does the lipase have to Serine proteases?

A

His, Ser and Asp are all in the same orientation

130
Q

What is tetrahydrolipstatin?

A

Potent phospholipase inhibitor used to treat obesity

131
Q

What structure do obesity inhibitors possess?

A

They have a lactone ring on them

132
Q

How can bacteria utilise lipases?

A
  • They can use them for bacterial warfare
  • They have to secrete the lipases into the target organism using type 6 secretion system which has a long tube with a spike that is injected into the target cell
  • The toxins travel through here and they can then cause damage
133
Q

Why would you not want an active lipase inside bacteria?

A

This could damage its own cells- protein deals with this by inhibiting the activity of the lipase to stop it working

134
Q

What is burkholderia cenocepacia?

A
  • Important pathogen that infects patients with cystic fibrosis
  • It secretes toxins using a type 4 secretion system, one of which is a lipase
135
Q

What are bacterial secretion systems?

A

Bacterial secretion systems are protein complexes present on the cell membranes of bacteria for secretion of substances

136
Q

How does B. cenocepacia protect itself from its own lipases?

A
  • It has an immunity protein which binds to and inhibits the lipase toxin
  • The immunity protein binds to the lipase by trapping the lid, preventing it from closing it on the active site to generate the active structure of the enzyme
137
Q

How is the lipase/immunity protein complex then activated?

A

The two molecules are secreted together from the bacteria and the disruption of this interface in the target cell leads to the activation of the lipase

138
Q

What reaction is catalysed by mandelate racemase?

A

The interconversion of R-mandelate to S-mandelate

139
Q

What is mandelate racemase a member of?

A

The enolase superfamily

140
Q

What metal cation does the reaction require?

A

The Mg2+ cation

141
Q

What is the mandelate racemase structure?

A

It has a chiral carbon meaning that it is interconvertible (two mirror images of it)

142
Q

What are the two areas of the mandelate racemase enzyme?

A
  • Barrel like beta strands: Where the structural activity occurs
  • Capping domain on the top
143
Q

What does the capping domain provide?

A

The capping domain provides elements of specificity

144
Q

What residues are involved in the catalytic activity in the beta strands?

A
  • Metal (magnesium is collated by an aspartate, a glutamate and another glutamate)
  • There is also another glutamate, a histidine and a lysine and all these residues are involved in general acid-base catalysis
145
Q

What do members of the enolase superfamily use?

A

They use an enzyme bound magnesium ion to facilitate catalysis

146
Q

What does substrate specificity arise from in the enolase enzyme?

A

Residues which lie at the interface of a capping domain and a barrel domain

147
Q

Where are residues involved in acid base catalysis found?

A

The barrel domain

148
Q

Where are residues found that bind substrate?

A

They are found in the interface of the capping and barrel domains

149
Q

What are the subfamilies of the enolase enzymes?

A
  • MLE subgroup

- MR subgroup

150
Q

What intermediate does the mandelate racemase reaction produce?

A

The reaction produces an aci-carboxylate intermediate which is stabilised by the metal ion (Mg2+)

151
Q

What does mandelate racemase use to pull off the proton from His?

A

Mandelate racemase uses a lysine (166) as a base to remove the L-proton and a His (297) as an acid to run a D-proton

152
Q

What does it mean if the enolase, MLE and MR amino acid sequences are different?

A

The fact the residues are not very conserved displays that the enzymes probably did evolve from the same ancestor but it was probably a long time ago

153
Q

What enzyme is enolase similar to in structure?

A

Methyl aspartate ammonia lyase

154
Q

What is the reaction of the methyl aspartate ammonia lyase enzyme?

A

It interconverts methylaspartate to mesaconate

155
Q

What is the role of a glycosidase?

A

Glycosidases hydrolyse sugars

156
Q

What is a lysozyme?

A

A lysozyme is an enzyme that is involved in the first line of defence against bacterial attack, that cleaves peptidoglycan and polysaccharide complex in cell walls of gram positive bacteria

157
Q

What do lysozymes have little effect on?

A

Gram negative bacteria

158
Q

Where is lysozyme found mainly in humans?

A

In bodily secretions such as tears and nasal mucus

159
Q

What family are lysozymes part of?

A

Glycosidases

160
Q

What is the structure of a gram positive bacteria?

A

Inner plasma membrane is surrounded by thick peptidoglycan cell wall

161
Q

What is the structure of a gram negative bacteria?

A

Inner plasma membrane surrounded by a thin peptidoglycan cell wall that is linked to lipoproteins in an outer membrane

162
Q

What makes up peptidoglycan?

A

Peptidoglycan is a repeating unit of poly NAG-NAM subunits

163
Q

What is NAM?

A

N-acetylmuramic acid

164
Q

What is NAG?

A

N-acetylglucosamine

165
Q

Where does lysozyme cleave in the poly NAM-NAG chain?

A
  • Lysozyme cleaves in between NAM and NAG in regions where there are not too many cross links
  • Lysozyme cleaves the NAM beta 1-4 NAG linkage
166
Q

How are peptidoglycan chains cross linked?

A

NAM beta (1-4) and NAG beta (1-4)

167
Q

What is the general structure of lysozyme?

A
  • Polypeptide chain 129 amino acids and four S-S bridges
  • Structure two lobes (domains) separated by a deep cleft
  • (left) a small beta sheet of mainly hydrophobic residues
  • (right) a hydrophobic core surrounded by short alpha helices
168
Q

What substrate binds into the lysozyme active cleft?

A

Substrate triNAG was observed binding to top half of cleft

169
Q

How many sugars does the lysozyme active site bind and how was this determined?

A
  • The active site will bind 6 sugars and this was determined as the rate of hydrolysis was highest at NAG x 6 and stayed the same as the NAG number increased
  • Therefore there are 6 subsites for how the enzyme reacts with its substrate
170
Q

What kind of interactions were there between triNAG and lysozyme active site cleft?

A
  • H bonds to oxygen and nitrogen atoms (on edges of sugars)

- Hydrophobic/vdw interactions with faces of sugars

171
Q

What are the catalytic groups in the lysozyme active site cleft?

A

Asp52 and Glu35

172
Q

What are the properties of Glu and Asp?

A

They both have carboxylic acid functions

173
Q

How would you expect Glu and Asp to be at lysozymes optimal pH~6?

A

Carboxylate ions

174
Q

How is glutamate actually at ph~6?

A

Glu is still uncharged (instead of O- it is actually OH) due to the hydrophobic environment

175
Q

What forms the covalent acyl enzyme intermediate in the lysozyme accepted mechanism?

A

A nucleophilic attack by Asp52 forms the covalent acyl intermediate

176
Q

How is the first product produced?

A

The Glu35 donates a protons and sugars diffuse away as the first product

177
Q

What enzymes break down sugars into smaller units?

A
  • Lysozymes (bacterial cell walls)
  • Lactases (lactose)
  • Amylases(starch)
  • Cellulases (break down cellulose —> glucose)
178
Q

What are neuraminidases used as?

A

Used by viruses and bacteria to penetrate cell walls (pathogenesis)

179
Q

How is starch hydrolysed?

A

Hydrolysis of alpha-1-4 glycosidic linkages

180
Q

What does starch break down into?

A

Starch + water –> maltose subunits

181
Q

What is the proposed mechanism for amylase?

A

Similar to that of lysozyme as amylase has an Asp and a Glu flanking sugar

182
Q

What created the tetrahdral acyl enzyme intermediate?

A

Nucleophilic attack by the Asp193 on the glycosidic bond

183
Q

How does Glu219 act in amylase?

A

It acts as an acid to donate a proton to the leaving group

184
Q

How is the reaction completed in amylase?

A
  • By a second step whereby water attacks which is activated as a nucleophile by removal of a proton from water by Glu219
  • Glu219 acts as a base to attack the acyl-enzyme intermediate
185
Q

What is lactase and what is its role?

A

Lactase is a glycoside hydrolase that catalyses the breakdown of lactose (disaccharide) into glucose and galactose

186
Q

What do mutations in lactase result in?

A

Lactose intolerance or abnormal expression

187
Q

What is cellulose cleaved into?

A

Glucose

188
Q

What is neuraminidase?

A

It is required for the release of virus form infected cells and for the spread go virus within the respiratory tract

189
Q

What does neuraminidase do?

A
  • Helps release virions from the host cell after replication and assembly
  • It does this by cleaving the sialic acid from host cell glycoproteins
190
Q

What is another name for sialic acid?

A

Neuraminic acid

191
Q

What is another name for neuraminidase?

A

Sialidase

192
Q

What are types of neuraminidase inhibitors?

A

Tamiflu and Relenza

193
Q

What do neuraminidase inhibitors do?

A

Block the active site of the enzyme resulting in viral aggregation at the host cell surface, reducing the number of viruses released from the infected cell

194
Q

What is the chemical equation for glucose dehydrogenase reaction?

A

Glucose + NAD(P)+ D-glucono-1,5-lactone + NAD(P)H + H+

195
Q

What did the structural determination of the glucose dehydrogenase enzyme reveal?

A
  • That the enzyme was a dimer
  • Enzyme uses NADP and NAD
  • Each subunit had two domains and a cleft in between
  • The substrate binds to the cleft whereby there is a metal ion cofactor
196
Q

What did experimentation reveal about the actual mechanism of glucose dehydrogenase?

A
  • That the actual mechanism was far more complicated than the textbook mechanism had suggested:
  • The water remains coordinated to the zinc
  • The ligands to the Zn change
  • The Zn and water move in the active site
197
Q

What allows for the collase of the alkoxide ion in the actual mechanism?

A

Movement of the zinc and changes in the coordination between the complexes provides an electrostatic driver to collapse of the alkoxide

198
Q

What are some features of good inhibitor: complementarity?

A
  • Van der waal forces; the vdw radius needs to be close enough or interaction but not too close as this will result in repulsion
  • Ionic interactions (eg COO- and NH3+)
  • Hydrophobic interactions
  • Hydrogen bonds (eg C=O—-H-N)
199
Q

What are hydrophobic interactions?

A
  • Hydrophobic interactions describe the relations between water and hydrophobes (low water-soluble molecules)
  • Hydrophobes are nonpolar molecules and usually have a long chain of carbons that do not interact with water molecules
  • Interaction between two non-polar molecules
200
Q

What are hydrophillic interactions?

A
  • When the substrate binds to water then that interaction is known as hydrophilic interaction and the contact angle between water and substrate will be very less
  • Molecules that have charged parts to them are attracted to the charges within the water molecule.
201
Q

What are some features of good inhibitor: mimicking the transition state?

A

Inhibitors are designed to share features of the transition state showing high affinity and no ‘strain’ is introduced when they bind

202
Q

Why does glucose dehydrogenase prefer NADP to NAD?

A
  • The enzyme prefers NADP to NAD as it has a lower Km

- The NADP cofactor also makes extra interactions with the phosphate group

203
Q

What other ion did the crystal structure display?

A

A sulfate ion

204
Q

When the two structures of enzyme bound NAD and NADP were overlapped, why did enzyme bound NAD structure seem to be in a slightly different position?

A

In the structure of the sulfate with NAD, there is an extra oxygen so it is displaced slightly to the right

205
Q

What is the reaction of D 2-hydroxyacid dehydrogenase from H. medditerranei?

A

2-ketoacid + NAD(P)H + H+ 2-hydroxyacid +NAD(P)+

206
Q

What complex structures CAN you observe without fast reaction?

A

Keto acid and and NAD (reduced coenzyme) and Hydroxy acid and NADH

207
Q

What is the productive complex?

A

If you use an inactive mutant

208
Q

What must you be aware of when looking at crystallography images?

A

That you have adapted the enzyme for studying it and you will get different bonding to what is naturally occurring

209
Q

What is no till agriculture?

A
  • Weeds are killed by a herbicide application

- Herbicide must be inactivated upon soil contact and seeds are sewn by drilling

210
Q

What are the benefits of no till agriculture?

A
  • Retains soil structure
  • Reduces soil erosion by run off
  • Reduces water loss by evaporation
  • Reduces energy costs
  • Increased carbon capture
211
Q

What is paraquat?

A
  • Generates destructive reactive oxygen species following electron donation by PSI
  • Contact inactivated by binding to silicates
212
Q

What is IGPD?

A

Imidazoleglycerol-phosphate dehydratase

213
Q

What is IGPD involved in?

A

It converts imidazole glycerol phosphate into imidazole acetate phosphate

214
Q

What metal ion cofactor does IGPD use?

A

Manganese

215
Q

What pathway is the IGPD enzyme involved in?

A

Histidine pathway

216
Q

What are enzymes chiral properties?

A

Enzymes are always chiral and often distinguish between two enantiomers of a chiral substrate

Biochemistry 2 (5 decks)

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