Autumn Term Flashcards
1
Q
What is the bond between nucleotides called?
A
N-glycosidic bond between the 1’ carbon and the base
2
Q
How does a phosphate bond with a pentose sugar?
A
The oxygen of the 3’ carbon will bind to the phosphorous of the phosphate
3
Q
Why do bases twist?
A
To maximise the overlap of pi orbitals
4
Q
What is different about the major groove?
A
It exposes more information and is easier to bind to
5
Q
What is base stacking?
A
DNA bases are flat with no 3D structure so they stack evenly
6
Q
Why is a single helix less stable than a double helix?
A
Because stacking is weaker in a single helix
More stacking means there is more overlap with adjacent pi orbitals and the freedom of movement of e-
Hydrogen bonds promote a double helix
7
Q
What forces do you need to remember?
A
van der Waals
8
Q
How does DNA respond to water?
A
DNA A = dehydrated form
DNA B = hydrated form
9
Q
Can DNA recover from denaturing?
A
Yes
10
Q
When is all DNA denatured?
A
90 degrees
11
Q
What does increasing the salt concentration do to DNA?
A
Neutralises the negative charge of the phosphate backbone and increases the base stacking by interacting with polar components of nitrogenous bases
12
Q
What proteins prevent DNA from re-annealing?
A
SSB proteins which straighten the DNA
13
Q
What does DNA helicase do?
A
Splits DNA using ATP
14
Q
What is the process of DNA replication?
A
Strands separated
Two pronged fork shaped molecule
DNA polymerase III can only go in one direction
DNA is looped around on the lagging strand to face the right direction
For the leading strand, the synthesis is continuous
The lagging strand loops for a short section (Okazaki fragments)
DNA polymerase III stops when it runs our of the loop and pushes the new strand away from the replication fork
Sliding clamp proteins stop DNA polymerase from sliding back
DNA ligase finishes the strand
SSB proteins stops the strand from bending back and annealing to sequences further back
15
Q
What is the purpose of DNA ligase?
A
To join nicks (gaps between nucleotides)
16
Q
Why is DNA polymerase I needed?
A
It removes RNA primers and replaces them with DNA
17
Q
Why are primers needed?
A
DNA polymerase can’t start without a primer
The RNA primer starts a new Okazaki fragment
18
Q
What is A-?
A
A conjugate base
An acid that has lost a proton
19
Q
What is HA?
A
An undissociated acid
20
Q
How do you work out [HA] in an acid base neutralisation?
A
Total acid - amount which is neutralised
21
Q
What is the total acid?
A
Undissociated and dissociated
22
Q
What are the three states that an amino acid can exist in?
A
NH3+
NH3+ and COO-
COO-
23
Q
What is the equation for Ka?
A
[H3O+] [A-] / [HA]
24
Q
What is the calculation for pKa?
A
pKa = -log 10 [Ka]
25
What is the Henderson Hasselbalch equation?
pH = pKa + log 10 [A-] / [HA]
26
What does it mean when pH = pKa?
The concentrations of the protonated and deprotonated species are equal
27
What does it mean pH < pKa?
[HA] > [A-]
28
What does a low pKa mean?
The amino acid will donate a proton
29
What is the equation for Keq?
Keq = [B] / [A]
30
What is the equation linking Vmax, Km, [S] and V?
V = Vmax [S] / Km + [S]
31
How do you calculate Km?
Km = (k2 + k1) / k1
32
What is the rate equation?
Rate = [A] [B]
33
What is the Linweaver burk plot?
1 / [S] plotted against 1 / V
Y intercept = 1 / Vmax
X intercept = -1 / Km
34
How do you relate V to the enzyme state concentration?
```
V = k2 [ES]
V = k2 [Etot]
Kcat = k2 = Vmax / Etot
```
35
What is the beer lambert law?
```
A = e c l
A = absorbance
e = extinction coefficient
l = length
```
36
How do you calculate the change in concentration from the change in absorbance?
/\ c / t = (/\ A / t) / e l
37
How do you get from mmol to μmol?
1mM = 1mmol / litre = 1μmol / ml
| Concentration to amount
38
What is the specific activity concentration?
Activity per mg of protein
Activity per μmol / min
Activity = μmol/min / enzyme mass (mg)
39
What is reaction velocity?
The enzyme reactivity
40
Does absorbance have units?
No
41
How do you convert enzyme activity in terms of absorbance change per minute to μmol / min?
Convert to mM / min using the extinction coefficient of the reaction product
Take into account the assay volume to change from concentration to amount
42
How does a competitive inhibitor affect Km and Vmax?
Km increases and Vmax doesn't change
43
What are serine proteases?
A family of enzymes that hydrolyse proteins and peptides
| The site contains a catalytic triad of three amino acid residues including a crucial Serine
44
What is required for catalysis in carbonic anhydrase?
Zn 2+
45
What does carbonic anhydrase do?
It catalyses the hydration of CO2
46
How does zinc help in the carbonic anhydrase reaction?
The zinc ion is able to bind to water
It is an equilibrium between water and a hydroxide form
Enables hydroxide to be present
Zinc stabilizes the formation of a hydroxide ion at a neutral pH
The OH is able to react with CO2
OH causes the formation of a bicarbonate ion
Adding water allows it to be released
When the bicarbonate ion is released, it forms carbonic acid
Zinc stabilises this because it is strongly acidic
Zinc has lowered the transition state because the pathway is now available
47
How does a fourth histidine help in the carbonic anhydrase reaction?
It is able to pick up protons and lose them
It sits in two positions
It is transmitting protons between the medium and the enzyme then delivering or extracting the proton from the active site
This one acid moves really fast to create a fast reaction rate
48
How is chymotrypsin specific?
It favours cleaving peptides on the carbonyl side of aromatic residues
The active site pocket is surrounded by hydrophobic residues and is large enough to accommodate aromatic residues
49
What does the active site of serine proteases contain?
Catalytic triad of three amino acid residues including a crucial Serine
50
Why is Serine made chemically active?
By the other residues of the catalytic triad
51
In an equation with A- and HA, what is the base and what is the acid?
```
A- = base
HA = acid
```
52
In an amino acid with a COOH or NH3 side chain, how will the pKa graph look different?
There will be three pKa values
53
What are the elements in a protonated amino group?
NH3+
54
Serine proteases
Not normally chemically active
Made reactive by other residues of the catalytic triad
Activation allows it to catalyse bond breakage
55
How is DNA proofread?
If the wrong nucleotide is incorporated, a hydrogen bond isn't going to be formed so the nucleotide will stick out farther or be closer than 1.1 nm
DNA polymerase stalls, goes from 3' to 5', activates exonuclease activity, cuts out the wrong base, corrects it
56
What is the condensed version of DNA?
Heterochromatin
Fewer genes
Genes aren't expressed
Found in areas of the molecule where stability is important e.g. telomeres, centromeres
Replicated late in the S phase so that the packaging problem can be overcome
57
What is the less condensed version of DNA called?
Euchromatin
| More genes which need to be accessed
58
How is DNA packaged?
DNA strand wrapped around histone molecules
Four histone proteins (2 copies of each) and 146 base pairs of DNA
Polypeptides = H2A, H2B, H3, H4
Linker DNA = DNA strand in between histone proteins which holds them together
59
How do you tell the difference between organic bases?
```
Adenine = two rings, nitrogen
Thymine = one ring, one double bond, CH3 group
Cytosine = one ring, two double bonds on the ring
Guanine = two rings, oxygen
Uracil = one ring, one double bond
```
60
Where is the organic base attached to the pentose sugar?
1'
61
What is the structure of RNA?
A right handed single helix
62
Does the peptide bond allow for rotation?
No
63
What is the CN distance?
1.32A
Single = 1.49A
Double = 1.27A
64
What is the Ramachandran plot?
The plot is used to show energetically favourable conformational regions of
protein structure
The axes show the polypeptide backbone dihedral or torsion angles- psi and phi
Alpha helices and beta sheets are found in the ‘allowed’ regions of the plot
65
What is the only amino acid that can form disulfide bonds?
Cysteine
66
Positively charge amino acids
Lysine
Arginine
Histidine
67
Negatively charged amino acids
Aspartic acid
| Glutamic acid
68
What is the van der Waals distance?
The contact distance where the attraction between the two charges is the strongest
Bringing two atoms too close together causes repulsion
69
How do water molecules arrange themselves around hydrophobic regions?
Water molecules arrange themselves around hydrophobic molecules to decrease the entropy of the system
Hydrophobic molecules group together to reduce the surface area and make them thermodynamically favourable
70
Membrane proteins
Integral proteins = span the layer
Peripheral membranes = associated with it
Membrane spanning proteins interact with the fatty acid tails
71
What are cofactors?
Small organic ompounds (coenzymes) or metals which are required to function properly
Protein - cofactor = apoenzyme
Protein + cofactor = holoenzyme
72
What are the cofactor modifications?
```
Phosphorylation: phosphate
Glycosylation: carbohydrate or sugar
Ubiquitylation: ubiquitin
SUMOylation: SUMO
Acetylation: COCH3 acetyl group
Hydroxylation: OH group
Methylation: CH3 group
```
73
Serine cofactors
Glycosylation: o linked, replaces OH
| Phosphorylation
74
Threonine cofactors
Glycosylation: o linked, replaces OH
| Phosphorylation
75
Asparagine cofactors
Glycosylation: added to CONH2
| Methylation
76
Proline cofactors
Hydroxylation: more rigid
77
Lysine cofactors
```
Hydroxylation
Acetylation: added to nitrogen
Methylation
Ubiquitination
SUMOylation
```
78
Phosphorylation
ADP + Pi = ATP
79
Lysine and histones
Lysine is positive so the histone proteins have positive tails
Adding acetyl to the lysine neutralises the charge
Causes it to have no affinity for DNA
80
Protein purification
Specific activity = number of enzyme units per milligram of protein
Purification factor
Purification may not be completed
Bacteria reproduce fast but may not be able to produce a human protein
Liquid chromatography
Ion exchange chromatography: cation exchangers
81
Alpha helix
Optimised hydrogen bonds between C=O (i) and NH (i+4) of another
Prolene is too rigid to form one
Overall dipole due to the N and C terminus
82
Beta pleated sheets
Parallel or antiparallel, joined by a series of turns and loops
83
X-ray crystallography
High sample concentration
Atoms will refract the rays
Can be used on any sized molecule
Atomic resolution
84
NMR
```
Sample must be dissolved in water
Only for small molecules
No crystallisation
Lots of peaks (lots of amino acids)
Closer to real protein structure
```
85
Cryo-electron microscopy
```
Protein in buffer solution
Freeze it on a grid
Look under a microscope
Larger molecules
Near atomic resolution
```
86
Stages of an enzyme catalyzed reaction
```
E + S
| k1
EST
| k2
E + P
```
87
What is the activation energy?
The energy needed to reach the transition state
88
What do enzymes do?
Lower the activation energy barrier
89
When is Vmax reached?
When all of the enzyme is present is in the form of ES and the enzyme becomes limiting
90
Is Km dependent on the production of ES?
Yes
91
What does a large Km suggest?
Weak binding
| Low affinity
92
Extreme lock and key
Very strong binding
Lots of energy needed
Transition state is less stable
Vmax and Km both decrease
93
Extreme induced fit
Very little energy given out
Favours transition state binding
High Vmax because less energy is required to reach the transition state
E does not bind to S very well so you need more [S] to reach V,ax
Increased Km
94
How is affinity and rate controlled?
They are balanced
95
What is Vmax in relation to turnover?
Vmax is the turnover number of substrate molecules converted into product by an enzyme when the enzyme is fully saturated with substrate
96
What is Kcat?
The number of moles of product generated per mole of enzyme per second
97
What do effectors do?
Regulate the enzyme, usually inhibitory
98
What is competitive inhibition?
Regulation by substrate analogues
99
What is non-competitive inhibition?
Regulation by binding at a distinct allosteric site
| Bringing about an effect from a distance
100
Adding more substrate to a competitive inhibitor
Increase the reaction rate
101
Adding more substrate to a non-competitive inhibitor
No effect because it will not affect the binding at the allosteric site
102
Aspartate transcarbamoylase
Binds two substrates
Eventually makes CTP
CTP inhibits the first step of the reaction as no more CTP needs to be produced
Catalysis by approximation (binding two substrates close together)
103
ATCase
Two subunits:
C = catalytic trimer
R = regulatory dimer, binds to CTP
CTP regulates by binding to a non-catalytic site via the reorganisation of the quaternary structure
Switches between the T and R states
T = tense, inactive, binds to CTP which readily inhibits activity, CTP binding stabilizes it and prevents the transition to an R state
R state = relaxed, active state, favours substrate over CTP
T and R exist in an equilibrium
ATP is an allosteric activator of ATCase because it stabilizes the R state, making it easier for the substrate to bind
S shaped curve on the Michaelis Menten graph
104
Keq equation
```
Keq = [B] / [A]
B = products
A = substrates
```
105
Km equation
Km = (k2 + k1) / k1
106
V, Vmax and Km equation
V = Vmax [S] / Km + [S]
107
At Vmax, ES = ...?
ES = Etot
108
Henderson Hasselbalch equation
pH = pKa + log 10 [A-] / [HA]
109
Beer-lambert law
A = e c l
| /\ c / t = (/\ A / t) / e l
110
Kcat equation
Kcat = k2 = Vmax / Etot
