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MIMS > Intro to Macromolecules, protein structure and enzyme catalysis > Flashcards

Intro to Macromolecules, protein structure and enzyme catalysis Flashcards

1
Q

why aren’t lipids true macromolecules?

A

the monomers aren’t covalently bound

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2
Q

what are sugars?

A

straight chain polyhydroxy alcohols with an aldehyde (aldose) or ketone (ketose) group

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3
Q

what is glucose?

A

a 6 carbon sugar with an aldehyde group at carbon 1

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4
Q

how many chiral centres does glucose have?

A

4 (C2, C3, C4, C5)

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5
Q

what enantiomers of glucose exist?

A

alpha and beta-ring enantiomers

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6
Q

what is the stable conformation of the glucose ring called?

A

the ‘chair’ conformation

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7
Q

how many possible chair conformations does beta glucose have?

A

2

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8
Q

at what position does fructose have a ketone group?

A

C2

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9
Q

which has fewer branches: starch or glycogen?

A

starch

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10
Q

what do cell-surface carbohydrates consist of?

A

a core pentasaccharide with additions

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11
Q

what do nucleotides consist of?

A

a sugar linked to 3 phosphate residues and a base

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12
Q

at what position is deoxyribose de-oxy?

A

the 2-position

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13
Q

what sort of sugar is deoxyribose?

A

an aldopentose

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14
Q

what end of a DNA strand are nucleotides added to?

A

3’ end

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15
Q

what is the difference between the 3’ end and 5’ end of DNA strands?

A

5’ end has free phosphate, 3’ end has free hydroxyl

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16
Q

which bases are purine?

A

adenine and guanine

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17
Q

which bases are pyrimidine?

A

thymine, uracil and cytosine

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18
Q

how many double stranded regions does tRNA have?

A

4

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19
Q

what is a ribozyme?

A

an RNA molecule that acts as an enzyme

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20
Q

what form is the chiral centre of amino acids in proteins?

A

L form

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21
Q

what form do amino acids exist in at neutral pH

A

Zwitterion form

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22
Q

why are unbound Cys side chains unusual?

A

they are often paired as covalent disulphide bonds

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23
Q

why is proline unusual?

A

side chain is covalently linked to N atom of amino group of amino acid- give rigid cyclic structure

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24
Q

why is the peptide bond a planar structure?

A

delocalisation of electrons from N onto the bond give it partial double bond characteristics so no rotation around bonds possible and bond length is between single and double (resonance hybrid bond)

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25
Q

what are the bonds in amino acids where rotation is possible?

A

link points- central C atoms of each residue and their bonds with N and C

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26
Q

what is the phi bond?

A

bond between central C and N in protein backbone

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27
Q

what is the psi bond?

A

bond between the central C and carboxyl C in protein backbone

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28
Q

what causes folding of the polypeptide chain?

A

rotation of phi and psi angles for each residue

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29
Q

what does the Ramachandran plot show?

A

combinations of angles found in protein structures

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30
Q

what is the alpha helix?

A

a single right-handed helix

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31
Q

what forms the alpha helix?

A

C=O oxygen on the peptide bond of 1 residues interacting with N-H hydrogen of residue 4 along the chain

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32
Q

what are 3(10) helices?

A

right handed helices where the H bonds are between i and i+3

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33
Q

what are the different properties of alpha and 3(10) helices?

A

alpha helices are less tightly coiled, more stable and more flexible

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34
Q

what is the secondary structure of proteins?

A

localised regular arrangement of polypeptide backbone formed due to H bonding between N-H and C=O groups of peptide bond

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35
Q

when are H bonds strongest?

A

when linear

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36
Q

what links secondary structure elements in polypeptide chains?

A

loop regions

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37
Q

what is a beta sheet?

A

strands of polypeptide chain that interact with neighbouring strands through N-H to C=O hydrogen bonding forming puckered sheets

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38
Q

which are more flexible: alpha helices or beta sheets?

A

beta sheets

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39
Q

what is the order of strength of protein interactions?

A

covalent bonds > H bonds = electrostatic interactions > Van der Waals bonds

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40
Q

what is the tertiary structure?

A

the overall 3D fold of protein

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41
Q

what determines tertiary structure?

A

side chains

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42
Q

when do hydrogen bonds form?

A

when H atom bonded to electronegative atom, so H has partial positive charge

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43
Q

what is the hydrophobic effect?

A

non-polar residues won’t form H bonds with water molecules, disrupts water-water H bonding so water molecules become ordered around hydrophobic side chains- hydrophobic groups cluster together

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44
Q

what are electrostatic interactions?

A

strong bonds formed between oppositely charged chemical groups

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45
Q

what is the difference in electrostatic interactions within hydrophobic cores vs in aqueous solution?

A

in aqueous solution the charges are shielded by interactions between water molecules, in hydrophobic core strength of interactions increases as no water

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46
Q

what are Van der Waals forces?

A

weak interactions between all atoms, occur as atoms can be transiently polarised by asymmetric electronic distribution

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47
Q

what are disulphide bridges?

A

disulphide bonds between neighbouring Cys residues as result of oxidation

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48
Q

why don’t disulphide bridges form within the cell cytosol?

A

they occur due to oxidation so don’t form in intracellular reducing environment

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49
Q

when will a protein stop folding into secondary structures?

A

when the polypeptide chain reaches a minimum energy conformation determined by maximising formation of these bonds

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50
Q

how many helices form a collagen chain?

A

3

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51
Q

what is the function of collagen?

A

provides strength to skin, bone, cartilage and tendons

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52
Q

is the collagen superhelix left or right handed?

A

left

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53
Q

what allows the helices in the collagen chain to pack together closely?

A

glycine residues in regular pattern

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54
Q

what causes tight bends in the collagen helices?

A

proline residues

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55
Q

what is the major driving force for folding of globular proteins?

A

the hydrophobic effect

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56
Q

how are strands of beta sheets generally depicted?

A

flat arrows pointing from N terminus to C terminus

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57
Q

how are alpha helices generally depicted?

A

as cylinders or helices

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58
Q

what are motifs/super-secondary structures?

A

commonly observed groups of secondary structural elements

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59
Q

what is the Βαβ motif held together by?

A

H bonds between strands and hydrophobic interactions between helix and strands

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60
Q

what does the βαβ motif often bind to?

A

DNA

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61
Q

what does an α loop α loop motif usually bind?

A

divalent cations

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62
Q

what are α helical hairpins held together by?

A

hydrophobic and ionic interactions

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63
Q

what are β sheets in Greek key motifs held together by?

A

H bonds between strands

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64
Q

what is a β meander?

A

antiparallel beta sheets that keep going

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65
Q

why is a proline residue required for a β hairpin turn?

A

proline has bend to backbone

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66
Q

what is a domain?

A

part of the protein chain that forms a compact unit with a globular cores, several motifs put together

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67
Q

what are multidomain proteins?

A

proteins with multiple domains formed from a single polypeptide chain

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68
Q

how many regions does functional insulin have?

A

2

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69
Q

which regions does functional insulin have?

A

A and B

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70
Q

what is the structure of preproinsulin?

A

linear structure, 3 regions (A, B, C)

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71
Q

why is insulin not a quaternary protein?

A

originally synthesised as a single linear chain

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72
Q

what is a homodimer?

A

a dimeric protein with 2 of the same polypeptides

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73
Q

what is a heterodimer?

A

a dimeric protein with 2 different polypeptides

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74
Q

what are antibodies?

A

proteins used by immune system for molecular recognition

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75
Q

what cells produce antibodies?

A

B lymphocyte cells

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76
Q

what is the antigen surface an antibody binds to?

A

the epitope

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77
Q

how many chains do antibodies have?

A

2 identical light chains, 2 identical heavy chains

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78
Q

what does the secondary structure of antibody heavy and light chains consist of?

A

4-stranded anti-parallel beta-sheet and 3-stranded anti-parallel beta-sheet stabilised by single disulphide bond

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79
Q

what are the domains in the light chains of antibodies?

A

1 constant domain and 1 N-terminal variable domain

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80
Q

what are the domains in the heavy chains of antibodies?

A

3 relatively conserved immunoglobulin domains, 1 N terminal variable domain

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81
Q

what are cofactors?

A

molecules used by proteins to provide chemical reactivity, not found in a.a. side chains

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82
Q

what will happen if cofactors are removed from the protein?

A

they are integral to structure so it will unfold

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83
Q

what is a co-substrate?

A

a molecule that is loosely bound and used once by a protein then released

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84
Q

what is a coenzyme?

A

a cofactor used in enzyme-catalysed reactions

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85
Q

what is the haem group of myoglobin/haemoglobin an example of?

A

a prosthetic group

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86
Q

what is a prosthetic group?

A

cofactor tightly attached to protein and used multiple times, integral to structure of protein

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87
Q

what is the function of myoglobin?

A

facilitates diffusion of oxygen around muscle tissue

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88
Q

what is myoglobin?

A

small monomeric protein found in vertebrate muscle

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89
Q

how many residues does myoglobin have?

A

153

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90
Q

how many alpha-helices does myoglobin have?

A

8

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91
Q

what is the (Fe)II ion at the centre of the myoglobin haem coordinated by?

A

4 N atoms, 1 histidine side chain, free 6th position to interact with O2

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92
Q

where is the haem group in myoglobin?

A

wedged in hydrophobic pocket

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93
Q

what is the function of haemoglobin?

A

carries oxygen in the blood, binds it in high O2 conc in lungs and releases in respiring tissues

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94
Q

how many subunits does haemoglobin have?

A

2 alpha-subunits, 2 beta-subunits

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95
Q

what does each subunit of haemoglobin resemble

A

myoglobin

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96
Q

what is the structure of each subunit of haemoglobin?

A

8 α-helices, buried haem group, binds oxygen in 6th coordination position

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97
Q

what are the oxygen binding properties of myoglobin?

A

simple hyperbolic binding curve

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98
Q

what are the oxygen binding properties of haemoglobin?

A

sigmoidal binding properties, 4 subunits bind cooperatively

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99
Q

how does cooperativity work in haemoglobin?

A

oxygen binding causes haem ring to adopt straight conformation with Fe(II) moving into ring centre. pulls His residue that coordinates Fe(II) towards oxygen moving α-helix which contains His, places strain on interface between subunits

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100
Q

what is the shared property of the external surface of membrane-spanning proteins?

A

hydrophobic

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101
Q

what does a hydrophilic internal surface of transmembrane proteins allow for?

A

recognition and transport of specific hydrophilic molecules

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102
Q

what is the membrane spanning part of most membrane proteins formed from?

A

alpha helices or beta barrels

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103
Q

how many identical subunits make up the potassium channel?

A

4

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104
Q

how many alpha helices do each subunit of the potassium channel have?

A

2

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105
Q

what allows the potassium channel to be embedded in the membrane?

A

external surface of the protein is hydrophobic

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106
Q

what allows the flow of ions through the potassium ion channel?

A

hydrophilic pore down centre of tetramer

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107
Q

what does the selectivity filter of the potassium channel do?

A

provides oxygen atoms from backbone C=O groups so ion can lose hydration water without loss of energy- and therefore can pass through the channel- doesn’t do this for Na+ as Na+ ion is smaller

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108
Q

how many K+ are allowed through the potassium channel for every Na+?

A

1000

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109
Q

what is the principle of X-ray crystallography?

A

protein crystals diffract X-rays, diffraction pattern can be used to calculate electron density which can be used to build model of overall 3D structure of protein

110
Q

what proteins is NMR spectroscopy an option for?

A

proteins smaller than 300 residues

111
Q

what happens in cryoelectron microscopy?

A

samples placed onto a grid and studied at very low temperatures. doesn’t require crystals

112
Q

what sort of techniques are used to separate a protein of interest based on size or charge?

A

fractionation techniques

113
Q

what is the simplest form of chromatography?

A

affinity chromatography

114
Q

what happens in affinity chromatography?

A

column material contains molecule that specifically binds to protein of interest. when sample passed through column and washed only protein of interest binds, others washed away

115
Q

how can the protein of interest be eluted from the column in affinity chromatography?

A

by adding competitive ligand

116
Q

what happens in ion exchange chromatography?

A

uses column with charged bead material- proteins bind to different degrees depending of charge. protein eluted from column using gradient of increasing cell concentration- salt disrupts electrostatic interactions between protein and column

117
Q

which proteins elute first in ion exchange?

A

loosely associated proteins elute first, most tightly associated require greater salt concentration to elute

118
Q

what is the outcome of gel filtration?

A

separates proteins by size

119
Q

what happens in gel filtration?

A

column resin contains a gel, as sample passes through column proteins can enter gel beads and be slowed by material. larger proteins pass around beads so move faster

120
Q

what determines structure of a protein?

A

its amino acid sequence

121
Q

what determines function of a protein?

A

its structure

122
Q

how can structure and function of unknown proteins be predicted?

A

comparing to known proteins as proteins with same structure often have similar sequences and functions

123
Q

what are zinc fingers?

A

simple domains found in DNA/RNA binding protein which can bind to DNA double helix

124
Q

what is the structure of the zinc finger?

A

zinc ion at base of finger coordinated by 4 amino acid residues- 2 His and 2 Cys

125
Q

where do zinc fingers insert into the DNA double helix?

A

in the major groove

126
Q

what do misfolded proteins often form?

A

amyloids- insoluble fibrous aggregates

127
Q

what diseases are caused by amyloids?

A

Alzheimer’s and prion diseases

128
Q

what happens in Alzheimer’s?

A

amyloids formed from 40-42 residue fragment from membrane protein in brain tissue

129
Q

what happens in prion diseases?

A

prion protein misfolds to form toxic filaments, misfolding occurs when PrP protein comes into contact with other misfolded PrP protein

130
Q

what are Bradford assays used for?

A

to determine total amount of protein present in a sample

131
Q

what dye is used in Bradford assays?

A

Coomassie Brilliant Blue

132
Q

what colour is Coomassie Brilliant Blue in acidic solution and when bound to protein?

A

green-brown in acidic solution, blue when bound to protein

133
Q

what happens in SDS Page?

A

protein sample heated in presence of SDS, which denatures it and gives it negative charge, sample then undergoes gel electrophoresis to separate proteins by size

134
Q

what is SDS?

A

a charged detergent

135
Q

what does ELISA stand for?

A

enzyme linked immunosorbent assay

136
Q

what happens in ELISA?

A

antibody that binds specifically to protein of interest is linked to solid support. protein will then bind to antibody, other protein molecules washed away. 2nd antibody linked to enzyme binds different site on immobilised protein. washed again. when chemical substrate added enzyme reacts to generate molecule that’s easily detected

137
Q

what do reactions catalysed by enzymes show?

A

greater rates, greater specificity (no side reactions), capacity for regulation

138
Q

how can enzymes make unfavourable reactions take place?

A

couple them to favourable reaction to provide driving force

139
Q

what is enthalpy?

A

heat

140
Q

why may a reaction need an enzyme?

A

due to thermodynamic or kinetic barrier

141
Q

what change in enthalpy is favourable?

A

negative (release in heat)

142
Q

what is entropy?

A

disorder

143
Q

what change in entropy is favourable?

A

becoming more disordered

144
Q

what is the Gibbs free energy equation?

A

change in enthalpy - temperature(change in entropy)

145
Q

what change in Gibbs free energy is favourable?

A

negative

146
Q

what formula relates equilibrium constant to change in Gibbs free energy?

A

∆G=RT(ln([B]/[A])- lnK)

147
Q

what is a favourable reaction, exergonic or endergonic?

A

exergonic

148
Q

what is standard free energy?

A

∆G=RTlnK, free energy for a reaction where starting point is clearly defined and each chemical species is initially present at 1M, pH is 7, temperature is 298K

149
Q

how do biological systems use ATP in reaction coupling?

A

hydrolysis of the phosphoanhydride bond of ATP releases energy, if coupled to unfavourable reaction then overall reaction can proceed

150
Q

how do enzymes reduce the kinetic barrier of reactions?

A

stabilise transition state, thus decreasing free energy of activation

151
Q

how do enzymes lower energy of transition state?

A

acid base catalysis, covalent catalysis, proximity effects, substrate strain

152
Q

what is the active site?

A

the location of an enzyme where a reaction takes place

153
Q

what properties must an enzyme active site have?

A

correct shape to bind the substrate, correct shape to stabilise transition state, allow product release by not binding product too tightly

154
Q

what is the lock and key model of enzyme activity?

A

early model, suggested active site of enzyme is rigid and complementary to substrate, substrate then fits into active site like key in lock

155
Q

what is the difference between the real model of enzyme binding (induced fit) and the lock and key model?

A

in real model binding is often flexible, as active site and substrate interact they become deformed- to become more complementary to each other

156
Q

what reaction is catalysed by carbonic anhydrase?

A

interconversion of CO2 + H2O <-> HCO3- + H+

157
Q

what does the active site of carbonic anhydrase contain?

A

an essential Zn ion coordinated in 3 positions by His side chains with a free 4th coordination position, a binding pocket for a CO2 molecule

158
Q

how do carbonic anhydrases work?

A

substrate water molecule occupies 4th coordination position of Zn ion, binding to Zn deforms the water, causes electron movement towards O atom. this polarisation weakens bonds within the water. another His residue is placed close to the substrate water and accepts a proton from it leaving OH- ion sttached to zinc. binding pocket for CO2 brings CO2 molecule into close proximity with OH-, electron rich O of OH- attacks positive carbon of CO2 resulting in formation of bicarbonate

159
Q

what are proteases?

A

enzymes that cut protein molecules by hydrolysis of peptide bonds

160
Q

what are examples of serine proteases?

A

thrombin, elastase, trypsin, chymotrypsin

161
Q

what is in the active site of serine proteases?

A

essential catalytic serine residue, charge relay system (His and Asp residues), specificity pocket, oxyanion hole

162
Q

what is the function of thrombin?

A

blood clotting

163
Q

what is the function of elastase, trypsin and chymotrypsin?

A

digestive enzymes

164
Q

what makes up the charge relay system in serine proteases?

A

a Ser residue, His residue and Asp residue which make up a catalytic triad

165
Q

what is the first stage in the reaction mechanism of serine proteases?

A

substrate binding: catalytic serine residue attacks carbonyl carbon of peptide bond forming covalent bond between substrate and enzyme. positive charge stabilised by His residue which is then stabilised by Asp residue

166
Q

what determines the substrate specificity of a serine protease?

A

specificity pocket, side chain of substrate peptide preceding peptide bond fits into specificity pocket

167
Q

what is a charge relay mechanism?

A

charges that develop during catalysis are stabilised by presence of neighbouring opposite charges

167
Q

what sort of peptides does trypsin select?

A

ones with positively charged Lys/Arg in R1 as these fit in specificity pocket

167
Q

what sort of peptides does chymotrypsin select?

A

ones with bulky hydrophobic residues (Phe, Trp, Tyr) in R1 as has large selectivity pocket

168
Q

what allows the active site of serine proteases to transiently stabilise the tetrahedral intermediate?

A

oxyanion hole

169
Q

what is the oxyanion hole?

A

a region of the active site of serine proteases which is well suited to bind to and stabilise a negatively charged oxygen ion through H bonding to backbone NH groups of Gly and Ser

170
Q

why does the oxyanion hole stabilise the intermediate and not the substrate in the serine protease mechanism?

A

carbonyl oxygen of substrate isn’t positioned to correctly enter the hole but formation of the tetrahedral intermediate distorts it placing negative oxygen in oxyanion hole

171
Q

what happens after the serine protease oxyanion hole stabilises the tetrahedral intermediate?

A

intermediate decomposes due to proton donation from His57, releases half of substrate as new peptide and remains attached to other 1/2 as acyl-enzyme intermediate

172
Q

what happens in stage 2 of the serine protease reaction mechanism?

A

active site regenerated, removing part of original substrate. water molecule enters active site, acts as nucleophile attacking carbonyl carbon of substrate peptide. charge relay and oxyanion hole stabilise 2nd tetrahedral intermediate. second part of substrate peptide released, enzyme regenerated

173
Q

what is the function of Asp proteases?

A

cut peptide bonds

174
Q

what is the difference between the mechanism of serine proteases and Asp proteases?

A

Asp proteases use pair of catalytic Asp side chains instead of reactive serine residue

175
Q

how do Asp proteases work?

A

one Asp (Asp1) begins reactive cycle in protonated state- donates proton to substrate to form transition state. Asp2 deprotonated at start of reaction cycle, accepts proton to stabilise positive charge in transition state (acts as base). Asp2 removes proton from water, activated water performs nucleophilic attack on C=O at peptide bond to be broken. C-N bond broken, N takes proton from Asp1

176
Q

how many people were living with AIDS in 2021?

A

around 38 million

177
Q

how many people died from AIDS in 2021?

A

over 500,000

178
Q

what are polyproteins?

A

multiple proteins linked covalently in inactive form

179
Q

what is the function of HIV protease?

A

cuts polyproteins to generate functional HIV molecules

180
Q

what sort of protein is HIV protease

A

an Asp protease

181
Q

what are ritonavir and saquinavir?

A

drugs which act as competitive inhibitors for HIV protease

182
Q

where does HIV protease cleave peptide bonds?

A

between bulky residues

183
Q

where is the active site of HIV protease?

A

at interface of the 2 chains that make up the molecule

184
Q

what are class 1 enzymes?

A

oxidoreductases

185
Q

what are oxidoreductases?

A

enzymes that catalyse oxidation and reduction

186
Q

what is a common type of oxidoreductase?

A

a dehydrogenase

187
Q

what is a dehydrogenase?

A

oxidoreductase which catalyses removal of electrons with a proton

188
Q

what is an alcohol dehydrogenase?

A

enzyme which removes hydrogen from alcohols forming aldehyde

189
Q

what are class 2 enzymes?

A

transferases

190
Q

what is a transferase?

A

enzyme which transfers a functional group from a donor molecule to an acceptor molecule

191
Q

what is a protein kinase?

A

a transferase that transfers a phosphate group onto a protein molecule

192
Q

what are class 3 enzymes?

A

hydrolases

193
Q

what is a hydrolase?

A

enzyme that catalyses hydrolytic cleavage reactions, breaking molecules by severing covalent bonds

194
Q

what is a protease?

A

a hydrolase which cuts a protein

195
Q

what is a DNase?

A

a hydrolase which cuts DNA

196
Q

what are class 4 enzymes?

A

lyases

197
Q

what is a lyase?

A

an enzyme which breaks covalent bonds by means other than hydrolysis or oxidation and creates new double bonds or rings in the process

198
Q

what is aldolase?

A

a lyase which breaks fructose-1,6-bisp to make 2 smaller molecules, generates C=O in the process

199
Q

what are class 5 enzymes?

A

isomerases

200
Q

what are isomerases?

A

enzymes which catalyse geometric changes- alter relative positions of chemical groups on molecule without altering groups present

201
Q

what are class 6 enzymes?

A

ligases

202
Q

what are ligases?

A

enzymes which join molecules together using ATP

203
Q

what do enzyme kinetics do?

A

analyse rate at which enzyme works and how rate varies depending on concentration of substrate + presence of regulatory molecules

204
Q

what are the uses of enzyme kinetics?

A

info about mechanism of action of enzyme, effect of different molecules on kinetics shows how enzyme is controlled in cell, quantify effectiveness of drugs, info about function in tissue

205
Q

what does hexokinase do?

A

converts glucose into glucose-6-phosphate, allows GluTs to take up glucose

206
Q

what does the different kinetics of glucokinase and hexokinase allow?

A

muscle to continue to take up glucose when blood sugar levels drop, liver to secrete glucose under these conditions

207
Q

what is the simplest form of enzyme kinetics?

A

Michaelis-Menten kinetics

208
Q

what does Michaelis-Menten enzymes do?

A

combine with substrate S to form non-covalent enzyme substrate complex which decomposes to form product

209
Q

what do Michaelis-Menten reactions assume?

A

there is no conversion of product back into substrate- only true during early stages of reaction when concentration of product very low so initial rates of reactions always measured

210
Q

what do rate constants do?

A

relate rate of reactions (v) to substrate concentration

211
Q

how is rate of production of product determined by k2 (rate constant for conversion of enzyme substrate complex to product) in Michaelis-Menten kinetics?

A

V=k2[ES]

211
Q

what relationship does the Michaelis-Menten equation describe?

A

relationship between initial rate of a reaction (v0) and initial substrate concentrations [S]

211
Q

what does derivation of the Michaelis-Menten equation assume?

A

fixed concentration of enzyme, fixed reaction conditions, enzyme conc. small compared to initial substrate conc

212
Q

what is the Michaelis-Menten equation?

A

v0= Vmax[S]/Km+[S]

213
Q

what does the relationship between V0 and [S] follow in Michaelis-Menten kinetics?

A

rectangular hyperbola

214
Q

what equation relates Vmax to enzyme concentration?

A

Vmax=k2[E]T

215
Q

what is [E]T?

A

total enzyme concentration for fixed enzyme concentration

216
Q

what is Vmax?

A

the maximum possible reaction rate, achieved when enzyme supplied with infinite substrate concentration

217
Q

what is Km?

A

substrate concentration when rate of reaction is half maximum rate

218
Q

what equation gives Km?

A

Km= (k-1 +k2)/k1

219
Q

what will the Km of an enzyme with a high affinity for its substrate be?

A

very low

220
Q

what is the gradient of a plot of 1/Vo against 1/[S]?

A

Km/Vmax

221
Q

what is the x-intercept of a plot of 1/Vo against 1/[S]?

A

-1/Km

222
Q

what is the y-intercept of a plot of 1/Vo against 1/[S]?

A

1/Vmax

223
Q

what do competitive inhibitors do?

A

bind directly to enzyme active site, compete with substrate for access- limit the time during which active site accessible to substrate so reduce rate of reaction

224
Q

what make the best competitive inhibtors?

A

transition state analogues, as active site optimised to bind to transition state

225
Q

what properties make HIV protease inhibitors good competitive inhibitors?

A

tetrahedral C residue attached to OH to mimic tetrahedral intermediate, lack N atom of peptide bond- prevents hydrolysis by the protease- so binds tightly and can’t be cleaved

226
Q

what is the effect of a competitive inhibitor on Vmax and Km?

A

Vmax doesn’t change, Km will increase as greater conc of substrate needed to reach half-maximal rate

227
Q

what is Vmax?

A

rate of reaction at infinite substrate concentration

228
Q

what is the effect of a non-competitive inhibitor on Vmax and Km?

A

won’t change Km, will decrease Vmax

229
Q

what do non-competitive inhibitors do?

A

alter conformation of important catalytic residue without altering amino acids that bind to the substrate

230
Q

what do allosteric inhibitors do?

A

alter shape of enzyme so active site less complementary to reaction transition state

231
Q

what is the effect of allosteric inhibitors on Vmax and Km?

A

decrease Vmax, increase Km

232
Q

how do nerve gases work?

A

irreversibly react by forming covalent bond with essential serine residue in active site of acetylcholine esterase which breaks down ACh- so muscle remains stimulated when nerve gases inhibit the enzyme- prevents breathing

233
Q

how does suicide inhibition work?

A

inhibitor resembles substrate but can’t go through complete enzyme-catalysed reaction. covalently binds to enzyme so enzyme can’t bind actual substrate- irreversible

234
Q

how does penicillin work?

A

suicide inhibitor. inhibits glycopeptide transpeptidase- essential for formation of peptide bonds in synthesis of bacterial cell wall

235
Q

why do suicide inhibitors make ideal drug molecules for targeting enzymes from pathogens?

A

have high specificity and are irreversible

236
Q

what is the most common form of reversible covalent modification at the active site?

A

addition of a phosphate group to enzyme

237
Q

which residues can be phosphorylated in the active site?

A

Ser, Tyr, Thr

238
Q

how does phosphorylation of the active site inhibit enzyme activity?

A

bulky phosphate group blocks substrate access

239
Q

how is Cdk2 controlled by phosphorylation?

A

phosphorylation of Tyr15 in active site of Cdk2 blocks binding of ATP into active site, prevents function of enzyme. removed when cell ready to divide

240
Q

how are zymogens activated?

A

part of N-terminal region cut away, so protein can undergo conformational change and active site can adopt high activity form

241
Q

what additional site do allosteric enzymes have?

A

site distinct from active site where allosteric effectors can bind

242
Q

how do allosteric activators work?

A

alter conformation of enzyme so active site is in better conformation to bind to/stabilise transition state

243
Q

what do mutations in glucokinase cause?

A

reduce activity of enzyme, reduce responsiveness of pancreas to glucose and thus insulin secretion

244
Q

how do allosteric activators work on glucokinase?

A

bind to site 20A away from active site, stabilise high energy conformation of glucokinase increasing its glucose sensitivity (decreases Km) and increasing rate of conversion to G-6-P (increases Vmax)

245
Q

what is the simplest case of sigmoidal kinetics?

A

binding of substrate causes enzyme to undergo conformational change from low activity conformation to high

246
Q

what is homotropic allostery?

A

when the substrate binding causes the conformational change

247
Q

what does homotropic allostery do?

A

facilitates control of enzyme function- small change in substrate conc can lead to large change in enzyme activity

248
Q

what is heterotropic allostery?

A

when molecules in addition to the substrate cause conformational changes to determine enzyme activity

249
Q

what are the kinetics of an allosteric enzyme at high activator concentration so all subunits in high activity form?

A

standard hyperbolic kinetics

250
Q

what happens to enzyme kinetics when allosteric inhibitor added to allosteric enzyme?

A

remain sigmoidal but higher substrate concentrations required for high activity

251
Q

what is phosphofructokinase (PFK)?

A

tetrameric enzyme in glycolytic pathway

252
Q

what reaction does PFK catalyse?

A

conversion of fructose-6-phosphate to frucose-1,6-bisphosphate

253
Q

effect of ATP of PFK pathway?

A

highly active when ATP concs low and reduced flux when ATP levels high

254
Q

what allosterically inhibits PFK?

A

ATP

255
Q

what allosterically activates PFK?

A

AMP

256
Q

what property is shown by the 4 subunits of PFK?

A

cooperativity

257
Q

what stabilises the high activity state of PFK?

A

fructose-6-phosphate- the substrate

258
Q

what reaction does glycogen phosphorylase catalyse?

A

first stage of breakdown of glycogen- forming glucose-1-phosphate

259
Q

what pathway does glucose-1-phosphate enter?

A

glycolysis

260
Q

properties of glycogen phosphorylase?

A

dimeric enzyme, show cooperativity

261
Q

difference between high and low activity forms of glycogen phosphorylase?

A

in high activity form active site is exposed to protein surface, in low active site is buried and inaccessible to substrate

262
Q

what allosterically inhibits glycogen phosphorylase?

A

ATP and glucose-6-phosphate (when these are abundant, glycogen breakdown not needed)

263
Q

what allosterically activates glycogen phosphorylase?

A

AMP

264
Q

effect of phosphorylation of glycogen phosphorylase?

A

activates enzyme stabilising high activity form.

265
Q

how is the phosphorylated and dephosphorylated form of glycogen phosphorylase differentiated?

A

glycogen phosphorylase a= phosphorylated, glycogen phosphorylase= dephosphorylated

266
Q

target of Tamiflu and Relenza?

A

influenza neuraminidase- catalyses reaction needed for release of mature virus proteins

267
Q

structure of Tamiflu to make it good inhibitor of influenza neuraminidase?

A

transition state analogue with additional chemical groups to occupy empty active site pockets + interact with active site side chains, positively charged group on 4 position to fill pocket in active site + form H bonds with 2 glutamate residues in this pocket, positively charged group made smaller, oxygen in ring exchanged for C to increase stability

MIMS (15 decks)

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