Proteins Flashcards
(239 cards)
1
Q
What are the three main questions biochemists ask?
A
How do cells sense and respond to their environments?
How do cells make and break molecules?
How do cells access and use the energy in food?
2
Q
Name the four interactions between amino acids that stabilise a protein’s tertiary structure.
A
Ionic bond (including metal ion coordination)
Hydrogen bond
Hydrophobic interaction
Disulfide bridge
3
Q
How do cells form polymers (in general)?
A
Dehydration reaction
4
Q
How do cells break polymers down (in general)?
A
Hydrolysis reaction
5
Q
Describe a protein.
A
A non-branching polymer of a specific sequence of amino acids joined by peptide bonds.
6
Q
How large are the macromolecules that proteins form?
A
50-100 Angstroms
7
Q
Name three methods of determining proteins.
A
Protein crystallography
Electron cryo-microscopy
NMR spectroscopy
8
Q
Give an example of a protein involved in cell signalling, and how it functions.
A
Insulin- signals cells to take up glucose
9
Q
Give two examples of proteins involved in digestion, and how they function.
A
Trypsin- breaks down proteins
Amylase- breaks down starch into sugars
10
Q
What does HIV Protease do?
A
Breaks down proteins in HIV viruses- essential for replication. Can be made non-functional by using a bound inhibitor.
11
Q
Give two examples of proteins involved in metabolism, and how they function.
A
Alcohol dehydrogenase- helps metabolise ethanol
Hexokinase- helps metabolise glucose by adding a phosphate group to keep it in the cell
12
Q
Give an example of a protein involved in oxygen transport, and how it functions.
A
Haemoglobin- binds oxygen in the lungs and carries it to tissues for use in metabolism
13
Q
How does SARS-CoV2 infect a host cell?
A
Spike protein unfolds and binds to the ACE2 receptor, which triggers fusion of the virus membrane with the epithelial cell membrane.
14
Q
What is the central carbon in the amino acid called?
A
Alpha carbon
15
Q
Which is the more predominant enantiomer of amino acid? How is it organised?
A
L-form
CORN (CO at left, R at top and N at right, with H pointing out of the page)
16
Q
How do amino acids exist in solution?
A
Zwitterionic form- the NH2 accepts a proton (NH3+) and the COOH loses a proton (COO-)
17
Q
Which part of an amino acid carries out biochemical reactions?
A
Side chain
18
Q
What is one way of classing amino acids into four groups?
A
Nonpolar amino acids (10)
Uncharged polar amino acids (4)
Negatively charged/ acidic amino acids (2)
Positively charged/ basic amino acids (3)
19
Q
What types of side chains do non-polar amino acids have?
A
- just hydrocarbon chains (aliphatic)
- ones containing sulfur
- rings with no polar group or available N attached
20
Q
What characteristics do aliphatic side chains give an amino acid, and how do they help it?
A
Oily ‘patch’
They will be buried inside a protein because they are hydrophobic- this stabilises protein structure.
21
Q
Name the amino acid with a side chain just consisting of hydrogen. What are some of its characteristics?
A
Glycine
It is non-chiral, and flexible because it’s small- so is commonly found in loops of a protein and in connective tissue.
22
Q
Where is cysteine often found and why?
A
In proteases, because it has a sulfhydryl group (SH) in its side chain which is often required for the metabolism of proteins.
23
Q
What is characteristic of phenylalanine and tryptophan amino acids?
A
They both have side chains containing aromatic rings- they are hydrophobic.
- bulky
- they have resonance, so ability to fluoresce
- used to determine concentration of proteins
24
Q
What is special about proline amino acid?
A
The side chain forms a closed ring, connecting to the amino group- this gives rigidity to protein.
Technically it’s an imino acid because it now contains a secondary amine.
25
What type of side chain do negatively charged/ acidic amino acids have? And why are they termed acidic?
One containing carboxyl group COO-
| They are displayed in their conjugate base form, so they were an acid that was deprotonated.
26
What type of side chain do positively charged/ basic amino acids have? And why are they termed basic?
One containing a positively charged amine (NH, NH2, NH3)
| They are displayed in their conjugate acid form, so they were a base that was protonated.
27
Where are charged amino acids situated in a protein?
On the surface, interacting with water- stabilises protein structure.
Or they might be tied up in a salt link with an oppositely-charged residue (electrostatic link).
Or it could be buried within the protein at the active site.
28
What type of side chain do uncharged polar amino acids have?
One containing a hydroxyl group (OH) or amide group (O=C-NH2)
29
What are one letter abbreviations of amino acids useful for?
Sequence alignment and mutations.
30
What does E6V mean, when describing a mutation?
There is a mutation of a glutamate to a valine at position 6 in the protein.
31
What enzyme is RT?
Reverse transcriptase, which helps replicate the viral genome in the HIV virus.
32
What is the pKa value for an ionisable group on an amino acid (or protein)?
The pH at which the ionisable group is 50% ionised (either protonated or deprotonated).
33
What is the pI of an amino acid (or protein)?
The pH at which the net charge is zero- zwitterionic form.
34
Arginine has a pKa of 12.5, what does this mean for its state at biological pH?
It is almost always ionised/ protonated.
35
Histidine has a pKa of 6, what does this mean for its state at biological pH?
Roughly half ionised, half not (close to 7.4)
36
What are the pKa's of the alpha carboxylic acid and amino groups?
2
| 9-10
37
What is the term for protein modification?
Post-translational modification
38
How do two (non-adjacent) cysteine amino acids bond?
In an oxidising environment, two hydrogens leave and a disulfide bond is formed.
39
Name six amino acid modifications.
```
Phosphorylation
Hydroxylation
Carboxylation
Metal binding
Iodination
Glycosylation
```
40
Describe phosphorylation in terms of proteins.
Adding a phosphate (top of side chain) to an enzyme can turn its activity on or off.
41
Which enzymes can phosphorylate other proteins? How does this work in the case of insulin?
Kinases, which are activated when insulin binds to the cell receptor. They activate proteins within the cell that are involved in glucose metabolism.
42
Describe what hydroxylation of proteins is good for, and which amino acids it involves.
Preventing connective tissue diseases and scurvy. Vitamin C is required for hydroxylation. Often proline and lysine are hydroxylated.
43
Describe what carboxylation of proteins is good for, and which amino acid it involves.
Blood clotting (essential), glutamate.
44
Which are the most commonly glycosylated amino acids?
Threonine and asparagine.
45
Why are proteins glycosylated?
Increase solubility
| Direct protein to correct binding unit
46
What are people measuring when they take blood sugar?
The amount of glycosylated haemoglobin (HbA1c).
47
What are the characteristics of the peptide bond?
Planar (due to 40% double character from resonance)
Trans
Partial dipole
48
What separates a peptide from a protein?
A protein is usually longer and has a defined biological function.
49
Define amino acid residue.
The term an amino acid is referred to as when it's been bonded with others in a peptide/ protein.
50
Around which atoms is a peptide chain flexible?
The alpha-carbons.
51
Most proteins are globular, what does this structure require of the peptide chain?
To double back and form a compact shape. Primarily composed of alpha-helices, beta-structure and turns.
52
What is the difference between secondary and tertiary protein structure?
Secondary= local 3D arrangement over a short stretch of ADJACENT residues
Tertiary= 3D structure of complete protein chain
53
What is the bond angle between N and alpha-C in the peptide chain?
phi Φ angle (anywhere between 0 to +/- 180 degrees)
54
What is the bond angle between alpha-C and C' in the peptide chain?
psi Ψ angle (anywhere between 0 to +/- 180 degrees)
55
What is the peptide bond angle between C' and N?
omega ω (180 degrees- trans, or 0 degrees- cis)
56
How can determining the bond angles of a protein help us as scientists?
We can use them to define its 3D structure.
57
When the peptide chain is shown as a flat zigzag, what are the main chain angles?
All 180 degrees.
58
Which type of collision can phi rotation lead to?
Oxygen to oxygen
59
Which type of collision can psi rotation lead to?
Amide to amide (NH to NH)
60
Why aren't all bond angles possible?
Steric crowding of Van der Waals nuclei
61
Name the bond angle and arrangement of a trans peptide bond.
ω of 180 degrees, alpha-carbons are on opposite sides of chain
62
Name the bond angle and arrangement of a cis peptide bond.
ω of 0 degrees, alpha-carbons are on same side of chain- steric hinderance is increased, so requires more energy to make, less common than trans
63
Describe the shape of an alpha-helix.
The main chain of the peptide spirals around a central axis (right-handed spiral- thumb= direction up, fingers= direction of chain).
64
What is the order of main chain atoms on a protein?
N, alpha-C, C'
65
How is an alpha-helix stabilised?
By hydrogen bonds- electrostatic interactions- between the slight positive charge on amide hydrogens and the slight negative charge on carbonyl oxygens.
They add 12-28kJ/mol stability.
66
What is the O-N distance in alpha-helices? Why don't we measure O-H?
~ 2.9 Å (four residues apart- O of n, H of n+4)
| Hydrogen atoms are too small to see in X-ray resolution.
67
How many residues per turn in an alpha-helix?
3.6
68
How far does an alpha-helix rise per turn?
5.4 Å
69
Where are the side chains in an alpha helix?
Pointing out of the helix.
70
What are the phi and psi angles of an alpha helix?
Φ ~ -57 degrees
| Ψ ~ -47 degrees
71
What are helix breakers?
Residues that end a helix structure.
Glycine- too flexible
Proline- too rigid, amide tied up in side chain, can't H-bond
72
In which direction is the alpha-helix dipole?
Positive at N-terminus, negative at C-terminus.
73
Define beta-strand.
A peptide strand with more extended structure than an alpha-helix. Unstable by itself- charged side chains. Typically contains 6 residues, but may have up to 15.
74
How is a beta-pleated sheet formed?
Forming hydrogen bonds between (2-10) adjacent beta-strands.
Makes a pleated sheet with right-handed twist.
75
Describe parallel and anti-parallel orientation of beta-sheets.
Parallel= N-C terminus directions of two beta-strands are the same. Hydrogen bonds are diagonal to strands.
Anti-parallel= N-C terminus directions of two beta-strands are the opposite. Hydrogen bonds are perpendicular to strands. (Lots of this in nature)
76
Which stretch of residues typically forms a beta-sheet ?
Non-polar, polar alternating. All non-polar side chains stick out above/ below, and all polar side chains stick out below/ above. They can form a structure with another similar sheet and create a non-polar region between them. e.g. silk
77
Describe a turn in protein structure.
- usually 3 or 4 residues where the strand changes direction
- require flexibility and rigidity- high glycine and proline content
- involve ~ 30% residues in a chain
- required to form globules
- commonly involves a hydrogen bond across the turn
78
Define random coil in protein structure.
A stretch of protein structure that doesn't fit into the standard groups (i.e. turns, helices, sheets).
79
Define protein domains.
Small proteins/ parts of proteins that can fold independently, and have a specific function within the protein. They can be reused and combined.
80
Define supersecondary protein structure.
Elements of secondary structure connected by turns or coils. (protein subdomains)
81
Name four common motifs of supersecondary protein structure.
Helix-turn-helix
Beta hairpin
Greek key
Strand-helix-strand
82
Give two examples of proteins that include helix-turn-helix supersecondary structure.
DNA binding proteins
| Calcium binding proteins
83
Describe beta-hairpin supersecondary structure and name two proteins that include it.
Two antiparallel beta-strands connected by a turn and H-bonds. Very common motif, of varying lengths.
Bovine pancreatic trypsin inhibitor
Snake venom toxin
84
Describe Greek key supersecondary structure.
Four antiparallel beta-strands- basically one long, bent over hairpin.
85
Describe strand-helix-strand supersecondary structure.
Beta-strand then a turn then a helix then a beta-strand. The strands are on the same plane (with the helix slightly above), so can stabilise each other via hydrogen bonds. Favourable interactions can occur between the side chains of the helix (pointing out) and the side chains of the strands (pointing up).
Very common motif.
86
Which component of a typical protein domain is very important for stability?
A hydrophobic core.
87
What are three families of tertiary structure that proteins can be grouped into?
Alpha domain family
Alpha/ beta family
Antiparallel beta family
88
Name two subgroups within the alpha domain family.
Four helix bundle
| Globin fold
89
Describe the four helix bundle tertiary structure.
Four alpha-helices tilted slightly to each other, in sequence. The tilting allows the side chains to pack more tightly. (helix, turn, helix, turn, helix, turn, helix, turn)
Hydrophobic core consists of oily residue side chains between the helices. The outsides of the helices have hydrophilic side chains. This means the helices are amphipathic.
90
Describe the globin fold tertiary structure.
Amphipathic helices with side-chains packed closely together within a hydrophobic core. But- unlike the four helix bundle- packing can occur between non-adjacent helices.
91
Name two subgroups within the alpha/beta family.
Alpha/ beta barrel
| Alpha/ beta horseshoe fold
92
Describe the alpha/ beta barrel structure.
Helix, turn, strand, turn, helix, turn, strand, turn, etc.
Strands circle inside helix circle and form a hydrophobic barrel core. Side chains at the top of the barrel are hydrophilic and point out into the solution- often the active site of an enzyme.
93
Describe the alpha/ beta horseshoe fold.
16 strand, helix motif repeats creating a horseshoe shape.
94
Name one subgroup within the anti-parallel beta family.
Anti-parallel beta barrel.
95
Describe the anti-parallel beta barrel structure, and one protein that has this structure.
Strand, turn, strand, turn etc.
Loop around to form a barrel with hydrophobic core. Present in antibodies and surface antigens.
Retinal binding protein is an anti-parallel beta structure. The retinal binds in the hydrophobic core, but has an OH hydrophilic group that sticks out the bottom.
96
What was biochemist Christian Afinsen's famous finding?
The amino acid sequence contains all the instructions required for the folding of the protein.
97
What is the likely sequence of events in protein folding?
(dictated by the need to build a hydrophobic core)
1. Short secondary structure segments form
2. Subdomains/ supersecondary structures form
3. Subdomains/ supersecondary structures come together to form a partly folded protein/ 'molten globule' that can rearrange
4. Final domain structure emerges
98
How do chaperone-dependent and chaperonin-dependent proteins fold?
Chaperone-dependent proteins require proteins that bind and assist temporarily (chaperones- e.g. Hsp70) to prevent the oily structures from aggregating.
Chaperonin-dependent proteins require chaperones, and a chaperonin protein (e.g. GroEL-GroES) that acts like a bin which the protein goes inside and ATP is used to fold it.
99
What are some examples of things that might denature a protein?
```
Change in pH (irreversible)
Heat (irreversible)
Detergents
Organic solvents
Urea
Guanidium
```
100
What is PrP, and what three conditions can it cause?
A prion protein that changes shape/ misfolds (alpha to beta), then promotes other proteins to misfold. This spreads and the prions aggregate to form plaques and tangles that destroy brain function.
Bovine spongiform encephalopathy (BSE)- eating beef that's contaminated with prions
Creutzfeld-Jacob disease (CJD)- receiving pituitary transplant of someone with prions
Kuru- ingesting human brain
101
Which two diseases do misfolded proteins (but not prions) contribute to?
Alzheimer's disease
Type 2 diabetes
Amyloid (type of misfolded protein) plaques can develop in the brain.
102
What is the concentration of haemoglobin in the blood?
~5 mmol/L
103
Describe the function of myoglobin.
Stores oxygen in muscles for use during intense exercise.
104
How do haemoglobin and myoglobin relate to evolution?
There is a strong evolutionary pressure for efficient oxygen delivery. The more oxygen you can get to your muscles, the faster and further you can run.
105
How much myoglobin do humans have?
0.5-0.7 mmol/L
106
Describe the structure of myoglobin.
Primary: ~150 amino acids
Secondary: 8 alpha-helices labelled A-H, connecting loops labelled AB, BC etc.
Tertiary: globin fold with a hydrophobic pocket, where a haem group is binded by His F8
Quaternary: monomeric
107
How are amino acids numbered in myoglobin and haemoglobin?
First, the letter of the helix they are present on, and second, their position within that helix. e.g. F8
108
Describe the structure of a haem.
A protoporphyrin, comprised of four pyrrole rings (of 4 carbons and a nitrogen) in a plane. Between the four nitrogens, the iron atom is held.
109
How is the haem bonded to the myoglobin?
The iron atom has six coordinate bonds to ligands:
- 4 to the nitrogen atoms of the haem
- 1 to histadine F8 of the globen
- 1 to O2 molecule
110
Why does the protoporphyrin (haem) have a red colour?
It has absorbance in the visible light spectrum because it has a huge molecular orbital due to conjugation between double bonds.
111
What will change the molecular orbital of haem, and what effect will this have? How can we see this effect?
Whether oxygen is bound or not (O is electron-rich), and the blood colour changes- duller red if no O2.
We can use spectroscopy- shine particular colour of light, and see if it absorbs it- allows us to identify compounds.
112
Why is the O2 bound by His E7 at an angle?
It is less stable in this position, which reduces the binding affinity of oxygen to myoglobin, so it's easier to release into the cell.
113
Why doesn't haem travel naked in the blood?
Without the histadine in the way, the oxygen would bind strongly to the iron atom, and would never be released.
114
Define allosteric control, and give an example involving myoglobin.
Controlling a protein's function 'without overlapping'.
Lactate (anion of lactic acid) decreases myoglobin's affinity for oxygen, but does not bind where oxygen binds. This promotes oxygen release from myoglobin.
115
What does the availability of O2 to cellular proteins depend on?
- pO2 in the local environment
| - binding affinity of O2 to haemoglobin
116
How do myoglobin and haemoglobin differ in O2 binding?
- myoglobin has a hyperbolic curve= only releases O2 (low saturation) when pO2 is very low
- haemoglobin has a sigmoidal curve, for cooperativity
Basically, myoglobin has tighter binding to oxygen, and haemoglobin has weaker binding.
117
How does pO2 vary throughout the body?
Much higher in the lungs (100 Torr), compared to the muscles (20 Torr). So haemoglobin can pick up a lot of oxygen in the lungs, and drop it off to the muscles.
118
Describe the shape of haemoglobin.
Tetramer of two alpha subunits and two beta subunits- all very similar to myoglobin. They interact non-covalently, and each contain a haem and bind one O2.
119
Describe the MWC model of cooperativity.
All four subunits of a tetramer must be in the same state- low-activity T tense, or high-activity R relaxed.
Binding each substrate (O2) shifts equilibrium in favour of R state- makes it more likely the tetramer will shift to the R state.
Explains sigmoidal curve of haemoglobin.
120
Describe the KNF sequential model for some enzymes.
There are intermediate forms where all four subunits don't have to be in the same state. Binding substrate makes binding the next easier (or harder- negative cooperativity).
121
Name three main differences between haemoglobin and myoglobin.
H transports O2, M stores O2.
H is a tetramer, M is a monomer.
M has a tighter-binding hyperbolic curve, H has a weaker-binding sigmoidal curve.
122
Summarise cooperativity in terms of haemoglobin.
Binding at one subunit makes binding at the successive subunits easier.
123
Why can't myoglobin show cooperativity?
It's a monomer- only one binding site.
124
What are the structural differences between deoxyhaemoglobin and haemoglobin?
In deoxyhaemoglobin, the F helix is risen, which pulls the haem upwards and giving it a dished shape. This shape is worse for binding O2.
In oxyhaemoglobin, O2 flattens the haem (pulls the Fe2+ into the plane of the haem), which pulls His F8 and helix F toward the binding site. This shape is better for binding O2.
125
Define a conformational change. What is an example in haemoglobin?
Shifts in the orientation of protein secondary elements.
Helix F moves relative to helix C.
126
How do the T and R states relate to types of haemoglobin?
Taut T-state = deoxyhaemoglobin
| Relaxed R-state = oxyhaemoglobin
127
How do the subunits of haemoglobin interact?
The F-helix of one haem interacts with the C-helix of another- moving from the T-state to the R-state, passing through the unstable intermediate state. Alternative interactions stabilise each state.
Moving into the R-state is its way of communicating it has bound an O2.
128
Describe BPG and its function within haemoglobin.
2,3-biphosphoglycerate is an allosteric inhibitor of O2 binding to haemoglobin. It is highly negatively charged, so can bind to the positive side chains (histadines and lysines) of the haemoglobin at the allosteric site- in the middle of the four subunits.
This electrostatic interaction stabilises the T state, reducing O2 affinity and promoting release of O2. BPG is produced during respiration in peripheral tissues when more O2 is needed.
129
What does the sigmoidal binding curve of haemoglobin tell us about its function at different points in the body?
The fraction of oxyhaemoglobin is very high at arterial pressure, so it is good at picking up lots of oxygen from the lungs.
The fraction of oxyhaemoglobin drops abruptly at venous pressure, demonstrating how cooperativity allows efficient unloading of O2.
130
Describe two other allosteric inhibitors of O2 binding to haemoglobin.
CO2- can bind to the extreme N terminal amino group
H+ (low pH)- can protonate certain amino acid side chains e.g. histidine
Both of these contribute to stabilising the deoxy-Hb conformation.
131
What occurs in haemoglobin at high altitudes?
Reduction in oxygen binding due to an increase in BPG. Allows oxygen to deliver more O2 to tissues.
132
How does foetal haemoglobin differ from adult haemoglobin?
It includes alternate isoforms, e.g. gamma, that have higher affinities for O2. This allows the foetus to capture O2 from the mother's blood across the placenta.
They also lack an amino acid at the BPG binding site, so bind BPG less well than adults.
133
At high altitudes, how do the mother and foetus continue delivering enough O2 to their tissues?
The mother's haemoglobin binding affinity drops significantly more than the foetus's, because the foetus can't bind BPG as well. BPG makes releasing O2 to the tissues easier.
134
Describe methaemoglobin.
Damaged haemoglobin in which the haem is oxidised from Fe2+ to Fe3+, which shifts one subunit to the R-state without having oxygen bound.
This keeps the other subunits in the R-state (cooperativity), so they don't release O2 into the tissues.
135
Describe Boston haemoglobin.
Haemoglobin with a H58Y mutation, or a His E7 mutation to Tyr E7. This causes Fe2+ to oxidise to Fe3+. The haem plane moves slightly, breaking the connection of Fe to His F8.
This stabilises the T-state, with low affinity for oxygen.
136
Describe sickle cell haemoglobin.
Haemoglobin with an E6V gain of function mutation. The valine binds really well to the hydrophobic pocket of another tetramer. These tetramers tack up and cause a deformed cell shape- can get stuck in capillaries.
137
Define enzymes and their function.
A biological molecule that catalyses (increases the rate of) a chemical reaction. Most enzymes are proteins- some are RNA's (ribozymes).
138
How do enzymes increase the rate of a reaction? What remains the same?
By lowering the activation energy. They don't change the free energy of products or reactants, and they don't change equilibrium.
139
Why do enzymes exist in the body?
To enhance specific chemical reactions in the cell and create organisation.
140
What does a negative Gibbs energy suggest?
Products dominate at equilibrium, energy is released, the reaction is favourable.
141
What does a positive Gibbs energy suggest?
Substrates dominate at equilibrium, energy is required, the reaction is unfavourable.
142
What does ΔG°‡ determine?
Activation energy (needed to reach transition state) determines the rate of reaction. The higher the activation energy, the slower the reaction.
143
What do aldolase and adenylate kinase have in common?
Enhance the reactions rates of unfavourable reactions.
144
Define isozymes.
Enzymes that catalyse the same reaction, but differ in sequence.
145
Define oxidoreductases.
A class of enzyme that catalyses redox reactions.
146
Define transferases.
A class of enzyme that catalyses reactions involving a transfer of a functional group.
147
Define hydrolases.
A class of enzyme that catalyses hydrolysis reactions.
148
Define lyases.
A class of enzyme that catalyses reactions that involve making/ breaking bonds without using H2O.
149
Define isomerases.
A class of enzyme that catalyses reactions that transfer atoms/groups within a molecule to produce an isomer.
150
Define ligases.
A class of enzyme that catalyses reactions that forms a bond between two molecules, usually coupled to ATP cleavage.
151
Name two ATP hydrolyases.
Myosin (in muscle), and ATP synthase.
152
Describe the properties of the active site on an enzyme.
- has amino acid side chains projecting into it
- binds the substrate via weak interactions
- determines the specificity of the reaction (how many substrates can it bind)
153
Name the types of enzyme-substrate bonds.
Ionic bonds- make use of charged side chains
Hydrogen bonds- good for specificity
van der Waals interactions- between any enzyme and substrate atoms in close proximity
Covalent bonds- rare, much stronger bonds
154
Describe the two models for enzyme-substrate binding.
Lock-and-key model (Fischer)- the active site is already the perfect fit for the substrate
Induced-fit model (Koshland)- the active site undergoes conformational change when it binds the substrate
155
Why are weak bonds used in enzyme-substrate binding?
They allow reversible binding- release of the substrate. If the bond is too strong, the Gibbs energy of enzyme-substrate complex is too low, and the activation energy is higher.
They also ensure specificity.
156
How do enzymes lower activation energy?
Ground state destabilisation
Transition state stabilisation- complementary to transition state
Alternate reaction pathway- lower energy transition state
157
Describe cofactors.
Can be organic (coenzymes) or inorganic (metal ions).
Position the substrate in the correct geometric and chemical orientation at the binding site of the enzyme.
Small molecules that carry an electron, atom or functional group.
Many coenzymes derived from vitamins (B).
158
Name 6 strategies by which enzymes drive catalysis.
```
Acid-base catalysis
Covalent catalysis
Redox and radical catalysis
Geometric effects
Stabilisation of transition state
Cofactors with activated groups
```
159
How many catalysis strategies do enzymes use?
Many use more than one.
160
What often drives redox catalysis?
Metal ions
161
Describe covalent catalysis.
Nucleophilic side chain of enzyme cleaves the substrate bond to form a reactive, short-lived intermediate, which is covalently attached to the enzyme. Another reaction- often hydrolysis- is required to remove the substrate from the enzyme.
162
Name two geometric effects that can catalyse a reaction.
Proximity and orientation.
163
Name four good nucleophiles.
RÖ:- (Hydroxyl group)
RS̈:- (Sulfhydryl group)
RN̈H2 (Amino group)
Imidazole group
164
What drives covalent catalysis?
Nucleophilic attack
165
What does almost every reaction that involves breaking a phosphate use?
Magnesium or manganese as a cofactor
166
How does Mg2+ catalyse the cleavage of a phosphate bond?
Establishes orientation of phosphates
| Stabilises negative charge on oxygens of two adjacent phosphates, making them better leaving groups and electrophiles.
167
What does acid-base catalysis involve?
Ionisable groups, in the correct ionisation state, and proton transfer.
168
Explain how enzyme activity is pH dependent.
Each enzyme has a characteristic optimal pH at which its rate is highest, because the amino acid side chains need to be in the correct ionisation state for the catalytic mechanism to proceed.
169
Which amino acid is particularly suitable to acid-base reactions, and why?
Histidine, because its side chain (an imidazole) has a pH of 6.5. This means it can easily accept and donate a proton at physiological pH.
170
Name some properties of chymotrypsin.
Since it's a protease:
- hydrolyase
- polypeptide and H2O substrates
- two shorter peptides or amino acids products
Chymotrypsin acts in digestion.
171
Name three serine proteases and how they are related.
Chymotrypsin
Trypsin
Elastase
Related through divergent evolution.
172
What is the difference between divergent and convergent evolution?
Divergent- have a common ancestor, and diverge (same structure, unique specificities)
Convergent- no common ancestor, but shared traits (different structure)
173
What two features do serine proteases share?
Catalytic triad- Aspartate, Histidine, Serine
| Specificity pocket- governs where the protein will be cleaved
174
What is the scissile bond of a protein, and how does the enzyme decide where to cut? Give examples for three serine proteases.
The bond to be cleaved.
The side chain next to the scissile bond sits in the specificity pocket of the enzyme.
Chymotrypsin- contains two glycines, so there's a lot of space for a large side chain
Trypsin- contains a negative aspartate, so only cleaves next to a positive side chain
Elastase- valine and threonine fill pocket, so only has room for small side chains
175
How can we tell subtilisin and chymotrypsin are related through convergent evolution?
Their peptide backbones do not superimpose- different structures. The same catalytic triad occurs, but not in the same order and structure.
176
How can we tell the catalytic triad is a very effective mechanism?
Evolution has found it through different ancestry routes.
177
Which catalytic mechanisms are used in chymotrypsin catalysis?
```
Geometric effects (proximity + orientation)
Covalent catalysis (nucleophilic attack + stabilisation of transition state)
Acid-base catalysis (moving a proton)
```
178
Where does the covalent bond from in chymotrypsin catalysis?
Between O of serine and alpha-C of peptide
179
What does a progress curve display? (enzyme)
The appearance of product/ disappearance of substrate over time.
180
What feature of an enzyme catalysed reaction do we usually measure?
The initial reaction velocity, Vi or Vo
181
How does the initial rate Vi change with increasing concentration of substrate [S]?
Increases linearly at first
As all the enzyme sites are occupied, the rate of reaction stops increasing- since we aren't increasing the number of enzymes, there's
182
Define Vmax on a V vs [S] curve.
Maximum possible velocity (when [S]=0. The asymptote that the curve approaches, but never reaches.
183
Define KM on a V vs [S] curve.
Michaeli's constant- the [S] at which V=Vmax/2
| The smaller the KM, the better the enzyme is at binding substrate.
184
Describe simple enzyme catalysis in terms of E, S, P, k1, k-1, and k2.
Enzyme, E, converts a substrate, S, into a product, P.
It first has to bind the substrate: enzyme-substrate complex.
k1 and k-1 are the rates of the forward and reverse reactions of the substrate binding/ releasing
k2 is the rate of catalysis- relates to activation energy required to reach transition state
185
Define the Michaelis-Menten equation.
V = (Vmax [S]) / (KM + [S])
| Describes the shape of the V vs. [S] curve
186
Name the five assumptions of the Michaelis-Menten equation.
- product is not converted back into substrate
- [S] >> [E]
- ES steady state (formation and breakdown rates of ES complex are equal)
- [S] does not change significantly (why we use initial V)
- all ES complexes have the same reaction rate
187
Which enzymes violate the Michaelis-Menten curve? Why do we need these?
Cooperative and allosteric enzymes. To act as control points in metabolic pathways.
188
How do cooperative enzymes violate the Michaelis-Menten curve?
Their V vs. [S] plot is sigmoidal, not hyperbolic, because cooperativity of subunits ensures they respond more steeply to intermediate changes in [S].
189
How does phosphofructokinase fulfill and violate the Michaelis-Menten curve?
With no allosteric inhibitors - hyperbolic curve. This happens at low ATP because we need to perform glycolysis to produce ATP.
With allosteric inhibitors (ATP or PEP) - sigmoidal curve. high ATP pushes enzyme into T state where it's much less active.
Cooperative behaviour
190
Describe the transition between T and R states in phosphofructokinase.
Positively charged Arg side chain binds substrate via electrostatic interaction. Loop flips to bring negatively charged Glu side chain into binding site, which repels the negatively charged substrate.
191
What does k-1 [ES] + k2 [ES} = k1 [E] [S] show?
Steady state assumption- that the rate of enzyme-substrate complex formation= rate of its breakdown
192
Describe the Lineweaver-Burk plot, and what the x and y intercepts mean.
The reciprocal of max velocity vs. the reciprocal of substrate concentration. (enzyme-catalysed reaction)
```
x-intercept= -1/KM
y-intercept= 1/Vmax
```
193
A more negative x-intercept on a Lineweaver-Burk plot shows what?
A large -1/KM = a small KM = high affinity for substrate.
194
KM = k-1 / k1
| Explain the formula and the assumption made.
KM describes the binding of substrate to enzyme. It depends on the formation of ES (k1) and the breakdown of ES (k-1 and k2).
Technically, KM = (k-1 + k2) / k1
But, since the breakdown of ES to form E + P (k2) is very slow compared to the equilibrium step between E + S and ES. Since the reaction is slow, k2 is very small, and negligible- k2 << k-1
195
How do enzymes maintain steady state in the body?
They will adjust their rate to keep their KM in the physiological range of [S].
196
When might an enzyme have two KMs?
When it can bind two different substrates.
197
Why does hexokinase have a hyperbolic V vs. [S] curve, while glucokinase has a sigmoidal V vs. [S] curve?
Hexokinase binds glucose to produce ATP. This is essential at low energy, so needs a fairly high rate with low glucose concentration.
Glucokinase binds glucose to produce starch. We don't want glucose being stored away at low glucose concentration, because we need it for energy, but we want to store it at high concentration.
198
Define kcat.
The turnover number= the number of substrate molecules converted to product per enzyme per unit of time (when E is saturated with substrate).
199
When does kcat = k2?
When the enzyme fits the Michaelis-Menten model.
200
How does kcat describe the rate limiting step? (formula)
Vmax = kcat {E}total
201
What does kcat / KM measure? Explain.
Enzyme efficiency
A high kcat and a low KM means a fast turnover and a low [S} for max velocity (high binding affinity).
202
Define the enzyme efficiency of a 'perfect catalyst'.
> 10^8 s^-1 M^-1
203
Give an example of a very efficient catalyst.
Acetylcholinesterase- breaks down neurotransmitter (ACh) is nerve synapses. Efficiency of 1.5 x 10^8
204
Name four reasons enzyme inhibitors are important.
Regulate metabolism
Basis of many drugs and poisons
Used to study enzyme mechanisms
Used to study metabolic pathways
205
Describe the classes and subclasses of enzyme inhibitors.
Irreversible= binds covalently to an aa side chain in the active site and deactivates the enzyme
Reversible= binds non-covalently to the enzyme and can be released, leaving the enzyme in its original condition
- competitive= inhibitor competes with substrate for binding at the active site
- non-competitive= inhibitor binds at a different site to the substrate (allosteric)
206
How does TPCK disable chymotrypsin?
End group covalently binds to the histidine of the catalytic triad, disabling it and filling the active site with its bulkiness. This blocks substrate binding.
207
What changes in competitive inhibition- Vmax or KM? Why?
KM increase, because more substrate is needed to get V up to half of Vmax (= KM).
Vmax doesn't change, because V can still be really high if [S] >> [inhibitor]. Most enzymes will bind the substrate instead of the inhibitor.
208
Define transition state analogues.
A class of competitive inhibitor that has the structure very similar to that of the transition state (between S and P). It will bind very well to the enzyme at the active site, but the reaction can't proceed- may be due to a bad leaving group.
209
When might a competitive inhibitor that proceeds to react be beneficial?
Alcohol dehydrogenase binds both methanol and ethanol. If [methanol] is too high, we can flood the space with ethanol to prevent poisonous formaldehyde from forming, and produce harmless acetaldehyde instead.
210
What changes in pure non-competitive inhibition- Vmax or KM? Why?
Vmax decreases, because they're not competing for the same site, so the inhibitor can decrease the rate.
KM doesn't change because the binding of inhibitor has no effect on the active site where the substrate binds.
211
Name the four common steps of activation and inhibition of proteins.
Chemical substance travels from its source.
Chemical substance binds to target protein.
Binding causes activation/ inhibition of protein.
Activation/ inhibition causes change in cellular response.
212
Define receptor.
A cellular protein (or assembly of proteins) that controls chemical signalling between and within cells.
213
How do receptors differ from enzymes?
```
Can have more than one binding site.
Bind ligands (not substrates).
Release the ligand unchanged (not converted to product).
```
214
Name the three classes of receptor.
Ligand-gated ion channel
G protein coupled receptor GPCR
Receptor tyrosine kinase RTK
215
Define ligand.
Chemical substance that specifically binds to a receptor.
216
Define endogenous and exogenous ligands.
Endogenous are produced in the body, exogenous are produced outside the body (drugs, poisons).
217
Where are most receptors found? What function do they serve there?
On the outer cell membrane, so they can sense changes in the extracellular environment and cause intracellular response.
218
Describe how ligands and receptors show specificity.
The size and shape of the ligand must match the corresponding receptor binding pocket. This allows enough chemical interactions for binding to occur.
219
How do medicinal chemists come up with new drugs?
They start with the chemical structure of an endogenous ligand, and modify it to make something safe and effective.
220
Define agonist and antagonist (ligands).
Agonist is a type of ligand that causes activation of a receptor when it binds.
Antagonist is a type of ligand that binds to a receptor and prevents the agonist from binding. This inhibits the receptor.
221
Describe signal transduction (receptor).
Receptor undergoes a conformational change, becoming active and starting a chain of events in which messages are passed through the cell.
222
Define second messengers.
Proteins or chemical signals that are free to move in the cell, so can pass on messages on signal transduction.
223
How do GPCRs start signal transduction?
Activate the G protein when agonist ligand binds.
224
Name two common G proteins, and their roles.
G alpha s (stimulatory protein that activates adenylate cyclase)
G alpha i (inhibitory protein that decreases the activity of adenylate cyclase)
225
How do RTKs start signal transduction?
By phosphorylating an adaptor protein when the agonist ligand binds.
226
Which enzymes perform phosphorylation in the cell, and how?
Protein kinases transfer phosphates from ATP to protein.
227
Which enzymes perform dephosphorylation in the cell, and how?
Protein phosphatases rapidly remove the phosphates from proteins to control signal transduction.
228
Define phosphorylation cascade.
A signal transduction pathway that uses many different protein kinases to phosphorylate/ activate the next in sequence, and ultimately activate the protein.
229
What occurs when a ligand-gated ion channel is activated?
Ions flow directly through the channel, into the cell, to produce effects. (no second messengers)
230
Name the key difference in performance of ligand-gated ion channels compared to GPCRs and RTKs.
Fast signalling
231
What are some factors that can change the response from a receptor?
Where on the cell/ body it is expressed.
Different relay molecules.
Pathway branching and cross-talk from other receptors on the cell.
232
What kind of ligand is insulin?
Endogenous peptide ligand.
233
On which three cell types does insulin act?
Muscle, adipose, liver.
234
How does insulin cause a response in muscle and adipose cells?
Activates RTK receptor, which phosphorylates an adaptor protein. This leads to further signal transduction, and finally the transport of GLUT-4 (a glucose transporter) to allow glucose entry into the cell.
235
How does insulin cause a response in liver cells?
Activates RTK receptor, which phosphorylates an adaptor protein. Further signal transduction leads to glycogen synthesis.
236
What kind of ligand is glucagon?
Endogenous peptide ligand.
237
How does glucagon cause a response in liver cells?
Activates GPCR, which activates a stimulatory G protein. Further signal transduction leads to glycogen breakdown.
238
Define GLP-1.
An endogenous peptide ligand (hormone) which is produced in the gut, and acts on pancreatic beta cells.
239
How does GLP-1 cause a response in pancreatic beta cells?
Activates GPCR, which activates a stimulatory G protein. Further signal transduction leads to insulin secretion.
