Protein biochemistry and enzymology! Flashcards
(141 cards)
1
Q
What is the Michealis-Menten equation?
A
v = Vmax [s] / Km + [s]
2
Q
What is the y-intercept on a lineweaver-Burke plot?
A
1/Vmax
3
Q
What is the x-intercept on a lineweaver-Burke plot?
A
-1/Km
4
Q
Define Km
A
Substrate concentration at which the rate is half of the Vmax - Michaelis constant
5
Q
What is VMax?
A
Maximum velocity
6
Q
Define Kcat
A
Turnover number - measure of how many substrate molecules an enzyme can convert into product per second when the enzyme is fully saturated with substrate
- number of products over time per second
7
Q
What is the equation to find Kcat?
A
Kcat = Vmax / [Etotal]
and so Vmax = kCat x [Etotal]
8
Q
What is [Etotal]?
A
total amount of enzymes added to reaction
9
Q
What happens to the Vmax and substrate concentration during competitive inhibition?
A
Vmax stays the same, and substrate concentration required to produce the Km is increased
10
Q
If inhibition is strong, is the Ki high or low?
A
Low - less inhibitor to half the Vmax, so the inhibitor is much stronger and more potent
11
Q
What is Ki and what does it measure?
A
Inhibition constant - measures how strongly an inhibitor binds to an enzyme. Represents the concentration of inhibitor needed to reduce the enzyme activity by half
12
Q
What does b-galactosidase hydrolyse other than lactose?
A
Nitrophenylgalactose
13
Q
What colour is the nitrophenolate ion?
A
Yellow
14
Q
At low concentration of substrate, how much product will you get?
A
Low amount of product because a low amount of enzymes bind
15
Q
At high concentration of substrate, how much product will you get?
A
High amount of product because a high amount of enzymes bind
- Enzyme optimum activity, works fastest it can
- start to get saturation
16
Q
What does increasing substrate concentration do to enzyme activity at already high substrate concentration?
A
- Increases substrate conc doesn’t make a difference because enzymes fully saturated
17
Q
What is affinity?
A
How much the enzyme likes the substrate, how attracted it is
18
Q
what are the 2 constants that describe an enzymes kinetic properties?
A
- Kcat (how fast an enzyme works - turnover number)
- Km (enzymes substrate concentration dependence)
19
Q
What is the specificity constant equation?
A
specificity constant = kcat/km
20
Q
What are the levels of kcat and km for a ‘good’ enzyme?
A
kcat = high and km = low
so then catalytic efficiency (specificity constant) is high because high/low is high
21
Q
What is the transient state?
A
Where a molecule is changing from one state to another
- it is very short lived
- In between two reactions
- there is an energy barrier and charge has to be stabilised
22
Q
What do binding surfaces do?
A
Stabilise tetrahedral intermediates and cause a charge relay
23
Q
What is a charge relay?
A
A charge relay system is a network of amino acids that relays charges (typically protons) in a coordinated way to activate a functional group for catalysis.
- typically serine proteases to attack peptide bond
24
Q
How can serine attack a peptide bond or carbon centre?
A
It has a hydrogen removed so it can be more electronegative and nucleophilic so can attack the carbon
25
What does enzyme inhibition mimic?
The transient state
- Lots of drugs are proteinase inhibitors e.g HIV drugs
26
What are the two types of inhibitors?
1. Reversible inhibitors
2. Irreversible inhibitors
27
What are reversible inhibitors?
competitive inhibitors
- able to bind to active site as well as substrate to block enzyme-substrate complex forming, can dissociate again
- reaches maximum velocity a lot slower
28
How do reverse inhibitors affect Vmax and Km?
- Vmax (maximum speed reaction can go) is the same but need a higher concentration of substrate to get to vmax
- So Km is higher
29
What is the equation of Km apparent?
Km app = km x (1+ [i]/Ki)
30
What is Km apparent?
measured in presence of inhibitor
- reflects Km with changes due to external factors
31
What happens to Km apparent in presence of reverse inhibitors?
Km app increases
32
What is Ki?
inhibitor constant
- measures how powerful an inhibitor is
- shows how tightly an inhibitor binds to an enzyme.
- used in drug development to assess how well a molecule inhibits a target enzyme.
33
What does a low Ki show?
stronger inhibition (higher binding affinity).
34
What does a high Ki show?
weaker inhibition
35
How do you determine Ki?
Measure rate without inhibitor and then rate with inhibitor
36
How do you determine -1/Km apparent on a graph?
Rate with inhibitor, -1/Km app is the x-intercept
37
What is an irreversible inhibitor?
A compounds that react covalently with groups within the enzyme that are essential to the enzymes activity
- Causes covalent modification of cysteine to inactivate enzyme permanently
-Sometimes it is reversible for a little while but when completely formed enzyme-inhibitor complex it cannot be reversed
38
What is the cysteine residue in an enzyme converted to by an irreversible inhibitor?
Iodoacetate
39
How do irreversible inhibitors affect Vmax and Km?
- Vmax changes but km does not
- can only get to a lower Vmax because fewer enzymes are available
40
What is Myasthaenia gravis?
An autoimmune disease,
- reduced signals at neuromuscular junction as start to block acetylcholine receptors by antibodies
- Causes progressive muscular weakness
41
How can Myasthaenia gravis be treated?
By raising concentration of acetylcholine
42
What is edrophonium?
A reversible (competitive) inhibitor of acetylcholinesterase
43
What is edrophonium used for?
To diagnose myasthenia Gravis but not so much to treat because of short half life
44
What is used to treat myasthenia Gravis?
- Neostigmine as it binds to acetylcholinesterase and makes a covalent bond so acetylcholine not broken down so much
45
What configuration do amino acids have in humans?
L -configuration because ribosomes only recognise L-amino acids
46
What is the process to turn L-amino acids into D-amino acids?
Racemization
- done by racemase enzymes
47
What is the R group of glycine?
H
48
What is the R group of alanine?
CH3
49
What is the R group of serine?
CH2OH
50
What is the R group of cysteine?
CH2SH
51
Is Nitrogen a good hydrogen acceptor or donor?
Good H-bond donor
52
Is Oxygen a good hydrogen acceptor or donor?
Good H-bond acceptor
53
When is an N-H-O hydrogen bond strongest?
When its linear
54
What are the secondary structures of proteins?
Alpha helix and Beta pleated sheets
- contain H bonds
- first set of folding
55
What is an alpha helix? Describe the structure
a right handed helix
- Has 3.6 residues per turn
- Good H bonding
- Side chains project off at 100 degrees to each other / to proceeding one
56
What are the links in an alpha helix?
n --> n + 4 link
This means that the carbonyl group (C=O) of the amino acid at position n will form a hydrogen bond with the amide group (N-H) of the amino acid located at position n + 4
This stabilises the alpha helix
57
What is an Alpha carbon?
C that connects amino group to carboxyl group
58
What are the 4 helix forming residues we need to know?
- Glutamate (E)
- Glutamine (Q)
- Alanine (A)
- Histidine (H)
Then also methionine, leucine, arginine, lysine
59
What is a helix destabiliser?
Tyrosine (Y)
Bulky side chain = steric hindrance
OH H bonding
Proximity etc.
60
What is a helix breaker?
Proline (P)
61
What does proline do to an alpha helix?
Its side chain binds to N on the alpha-carbon so prevents the N-alpha-carbon bond rotating
- breaks the helix
62
What are anti-parallel beta sheets?
Sheets are in opposite directions to each other
- they can H bond between the H and Os
- the sheets are formed from beta strands
- have straight N-H-O bonding (v strong)
63
What are anti-parallel beta sheets used for?
Mechanical support like in silk
64
What is the structure and properties of silk?
Made of protein fibroin
- has repeating glycine and alanine
- Silk won't stretch longitudinally but is very flexible laterally
- Strong in one direction, bendy in another --> property of beta sheet
65
Why is silk resistant to longitudinal stretching?
The backbone chain is already extended to a beta strand
66
What are parallel beta sheets?
Sheets go in same direction
- N - H -O bonding not as straight so less strong
- so H bonds not as straight, go diagonally
67
Are side chains involved in secondary structure?
No only backbone is involved in secondary
Structure
68
What are the three structural motifs? Examples please ladies
1. Alpha e.g myoglobin
2. Beta motifs ( also called beta turns or hair pins) e.g antibodies like immunoglobulins
3. Alpha-beta motifs e.g triose phosphate isomerase (TIMs!!!!) where there is an alpha/beta barrel
69
What are alpha-beta motifs?
strand and helix and comes back into another strand to form beta sheet
70
Why do proteins fold into tertiary structure?
due to hydrophobic effect of side chains
71
What is hydrophobic collapse?
an Entropic effect
- makes the protein fold initially
- Hydrophobic molecule makes water molecules form a ‘cage’
- so water molecules are more ordered than if hydrophobic molecule wasn't there
- like in oil and water
72
What are some hydrophobic side chains?
Valine, leucine, phenylalanine
- very long penis
73
What are some hydrophilic side chains?
Apspartate, lysine, serine
- Amazing long sex
74
What forces/bonds are there in tertiary protein structure?
Van der Waals interactions
Hydrogen bonding
Disulphide Bridges
Ion pair bridges (hydrophilic and hydrophobic interactions)
75
What are disulphide bridges?
Strongest bond
- Covalent bonds between 2 cysteine side-chains
- Oxidization to form this bond
- More common in extracellular enzymes than intracellular
76
What is an ion pair bridge?
A salt bridge
- draws oppositely charged side chains together
- important in stabilisation of 3D structure
77
What is the equation for pH?
pH = -log10 [H+]
78
What is the equation of [H+]?
[H+] = 10^−pH
79
What is pKa?
pKa is the pH where the acid-base pair is 50% ionized/dissociated
Higher pKa, weaker acid
80
What is the equation for pKa?
pKa = log10 (Ka)
81
What is the Henderson-Hasselbach Equation?
pH = pKa + log10 ([base]/[acid])
82
What is a base?
Proton acceptor
83
What is an acid?
Proton donor
84
What is a domain? and an example?
A globular unit formed from part of a polypeptide often associated with particular function
e.g yeast hexokinase
85
How are domains formed?
Often formed and coded independently by different exons in the gene
86
What does a large domain bind?
ATP, glucose binds in between
87
How can domain movement be driven?
By the binding and hydrolysis of dGTP as some enzymes have dGTPase
88
What is the quaternary structure?
Assembly of more than one polypeptide chain
89
4 examples of quaternary proteins?
1. Haemoglobin
- it is a tetramer as it has 2 alpha chains and 2 beta chains - alphaglobin and betaglobin
2. RNA polymerase
3. Collagen
4. HIV proteinase dimer
90
Describe collagen
Most abundant protein in body - ¼ of protein in human body
- 3 stranded coil
- High proption of proline and modified prolines to hydroxyproline
- Has post-translational modifications
91
Describe HIV proteinase dimer
- Encodes proteinase essential for maturation (poly protein processing) of new viruses
- Essential for assembling new HIV virus through smaller pieces
(like plastic model kits/airfix kits where you have all the little pieces together than you have to cut off so you can assemble them e.g a little car kit)
92
What drugs are used against HIV?
Protease inhibitors to block the HIV proteinase dimer enzyme and slow progression of HIV
93
What is the function of protein folding?
Structure and activity
Necessary for drug design
Necessary for protein engineering
94
SO what is a major driver of protein folding?
Hydrophobic effect and collapse
95
What does it mean if pH and pKa are the same?
50% of side chain is dissociated
50% acid, 50% base
96
What is protein purification?
Separation of protein you want from all others in the preparation
- recognition and then an assay
97
What is SDS page?
A type of gel electrophoresis
- Sodium Dodecyl Sulphate: a detergent
and PAGE is PolyAcrylamide Gel Electrophoresis
98
How does SDS work?
It unfolds the protein by coating the unfolded chain
- used to separate proteins based on molecular weight
99
What are the steps in SDS PAGE?
1. Boil the sample to overcome Van der wall forces and H bonds and denature it
2. Use reducing agents to break disulphide bonds
3. Proteins become coated with SDS
100
What is the SDS PAGE gel stained with?
Coomassie blue or silver nitrate
101
Why are marker proteins included in SDS PAGE?
Inclusion of marker proteins of known molecular mass allows molecular mass of other proteins to be estimated
102
Why is SDS PAGE considered an analytical technique?
Because protein is denatured
- not useful for purification those but useful for analytics
103
What do you do once completing SDS electrophoresis?
Plot a graph
Log of molecular mass in kDa (x-axis) against relative mobility
104
What does SDS calculate?
Sizes of protein in denatured state
105
Proteins have different structures which leads to different what?
Different properties
- can be separated based off different properties
- like in a game of Guess Who
106
What is separation by solubility?
Separating proteins based off the surface of soluble protein
- soluble proteins feature many polar residues which causes their surface to differ
107
What is precipitation by ammonium sulphate?
Salting out
- Depends on polarity of protein surface
- This determines how soluble it is in water
108
Explain salting out
Add ammonium sulphate and dissolve protein
Centrifuge
Pour supernatant into a new tube
Redissolve the pellet for presence of protein
Analyse the 2 fractions: one soluble at that concentration of ammonium sulphate and one not
109
What does adding ammonium sulphate do in protein separation?
Decreases water available to precipitate the protein
Basically uses up all the water so proteins can't bind to the water and they end up sticking together and forming a precipitate instead which can be centrifuged
110
If the protein is still in solution and not precipitated, what should you do?
Use a higher concentration of ammonium sulphate
111
What is a benefit of ammonium sulphate?
It doesn't denature the protein, just precipitates it so you can carry on using it
It also really likes water so uses all of it up
112
What is isoelectric focus point (pI)?
pH where a molecule has no net charge, is charged but no overall charge on molecule because charges balance out
113
What is isoelectric focusing?
1. Immobilised pH gradient strip and an electric field used and gel stained with Coomassie Blue or silver nitrate
2. Protein mixture is applied
3. Proteins move along pH gradient (if -ve go to +ve electrode which is at lower pH)
4. Charges on the protein are reduced until you get to the isoelectric focus point (pI) where there is a net zero charge and they stop moving
5. The electric field has no effect
114
Does the electric field have an effect when protein reach their isoelectric point?
No, they stop moving and have no net charge
115
What is isoelectric focusing used for?
Analytic techniques - separates proteins based on isoelectric point
- there's a limit to how much protein you can load though
116
What is 2 dimensional gel?
Gel electrophoresis that combines SDS PAGE and isoelectric focusing to separate proteins based on molecular weight and isoelectric point
117
What is Column chromatography?
A matrix in a Colum to separate proteins based off different properties
118
How can proteins be detected?
By absorption of UV light at 280nm
- can't be detected by colour bc most protein is colourless
119
Why do proteins absorb UV light at 280nm?
Because of aromatic amino acid residues like tyrosine and tryptophan
120
What is size exclusion chromatography (SEC)?
Gel filtration and molecular sieving
- it is a column with beads in that have lots of cross linked molecules and pockets in
- proteins fall through the beads in the column depending on size
121
What does size exclusion chromatography (SEC) separate off of?
Separates proteins according to size
- bigger proteins separated off first and then middle sized and then smaller proteins
122
How does size exclusion chromatography work?
Small proteins can enter the beads freely and lodge there and then move onto another bead but don't fall fast
Middle sized proteins can only just fit into the bead pockets so don't stay there long and fall (elute) through column
Big proteins are too big to fit in bead pockets so just fall down the column
- like a cattle grid, small kids will fall through the holes but bigger adults just pass over it
123
Benefits of size exclusion chromatography?
Good for purification as proteins not denatured so reflects natural state of the protein
Can purify complexes rather than single chains
Can calibrate the column with known proteins and seeing how long it takes them to fall through the column to then make a standard curve
124
How can you estimate molecular mass by SEC?
Using the calibration curve to workout any unknown protein mass (elution volume) and structure
125
How can you combine SDS PAGE and SEC?
SDS PAGE gives the monomer size and SEC gives the native size - so can workout if its a dimer in its natural state
These indicate its quaternary structure!!!
126
What is ion exchange chromatography?
Depends on ionisable groups in protein (pH is important)
Charged groups on protein interact with charged groups on the column
Cation exchange reacts with cations in a -ve column
Anion exchange reacts with anions in a +ve column
127
What is affinity chromatography?
Used for purification based on proteins having a selective affinity for a particular structure
- enzyme complementary to a specific substrate
128
How does affinity chromatography work?
1. Ligands are immobilised (e.g substrate or inhibitor) on a column
2. Solution of proteins are washed through the column and the specific protein of interest binds to the specific ligands used and the rest of the proteins move through the column
3. The bound protein of interest is then eluted through the column by disrupting the binding and interaction
129
How can protein elution be achieved in affinity chromatography?
By changing the pH, solute concentration or a competing ligand
130
How can tags be used in affinity chromatography?
Can take a gene and express it as a protein and add a tag which will also be translated - recombinant protein
Can then use the properties of tags to purify the protein
131
3 examples of proteins with tags used in affinity chromatography?
Maltose binding protein, glutathione-S-transferase, (His)6 tag
132
What is the His-tag on a protein?
Can interact with nickel to immobilize protein on column
- can change the pH or add a purifying soluble chemical to displace histidine to bind to nickel so the protein is eluted
133
What is the Kcat, Km and Kcat/Km for a 'good' enzyme?
Kcat high
Km low
Kcat/Km high
134
What is Myasthaenia Gravis?
Progressive muscular weakness
Problem is at neuromuscular junction
Acetylcholine receptors become progressively blocked by antibodies
(Autoimmune disease)
Condition can be relieved by raising
concentration of acetylcholine
135
What is Edrophonium?
A competitive inhibitor of acetylcholinesterase
Used in diagnosis of Myasthaenia Gravis to increase acetylcholine
NON-COVALENT, REVERSIBLE
136
What is neostigmine?
Competitive reversible inhibitor of acetylcholinesterase
Used in treatment of Myasthaenia Gravis to increase acetylcholine
137
What is a zwitterion?
A zwitterion is a molecule that contains both a positive and a negative charge, but has an overall neutral charge.
Zwitterions have at least two ionizable groups, one of which carries a positive charge (typically a amine group, -NH₃⁺) and the other carries a negative charge (typically a carboxyl group, -COO⁻)
Amino acids are one example
138
What is the alpha carbon of an amino acid?
The carbon bonded to:
- Carboxyl group
- Amine group
- Hydrogen
- R group
139
What bonds join proteins?
Peptide bonds
140
What is the hydrophobic effect in protein tertiary folding?
The hydrophobic effect is a major driving force in protein folding, causing hydrophobic amino acids to cluster in the protein's interior, away from water
141
How do you find pKa?
pKa = -logKa
Same with pKb and Kb
