Proteins Flashcards
(158 cards)
1
Q
What does the structure of a protein tell us?
A
Its function
2
Q
Suffix -ase in proteins means? What does the word in front mean
A
Its an enzyme. Word infront tells what happens
3
Q
Example of cell signalling protein
A
Hormone insulin
4
Q
Examples of digestion proteins
A
Trypsin, HIV protease, amylase
5
Q
Examples of metabolism proteins
A
Alcohol dehydrogenase, 1CZA hexokinase
6
Q
Example of oxygen transport metabolism protein
A
Hemoglobin
7
Q
Metabolism and energetics membrane protein
A
ATP synthase
8
Q
Immune protection protein
A
Antibody
9
Q
Replication and maintenance proteins
A
DNA polymerase, RNA polymerase
10
Q
What are the different properties of amino acids
A
Non-polar
Polar and negatively charged
Polar and positively charged
Polar and uncharged
11
Q
Meaning of pKa and pI
A
pKa- where group is 50% ionised
pI- pH where net charge = 0
Both linked to when amino acid is in zwiterionic form
12
Q
Ways which post-translational modification can occur
A
- disulfide bond formation cystein-cystein. Reversible
- phosphorylation
- hydroxylation
- carboxylation
- glycosylation
- metal binding
- iodination
13
Q
Features of the peptide bond
A
Bonds amino acids, is planar, trans and has dipoles
14
Q
What are amino acids referred to when in a peptide chain and why?
A
Amino acid residues because they are no longer complete individual amino acids.
15
Q
What is the order in which a protein runs
A
N-C-CO-N-C-CO
16
Q
What are the levels of protein structure?
A
Primary, secondary, tertiary and quaternary
17
Q
How do proteins fold and what are the different types
A
Fold via flexible bonds which rotate atoms around them. There are phi bonds (N-C), psi bonds (C-CO) and an omega bond (N-CO) which isnt very flexible and barely moves
18
Q
Why is the omega bond not flexible
A
Due to rigidity and resonance
19
Q
How far can phi and psi bonds move
A
0 to +/- 180
20
Q
What would happen if phi and psi bonds were unrestricted
A
Phi- O-O collision
Psi- NH-OC collision
21
Q
What two forms can omega bonds be
A
Cis (0°) or trans (180°)
22
Q
Two types of secondary structures
A
Alpha helix and beta structures
23
Q
Features of an alpha helix (6)
A
- main chain spirals to the right hand side around a central axis
- hydrogen bonds occur from CO-HN ~2.9Å stabilising the structure
- side chains point out from the helix to stabilise structure
- phi ~-57° and psi ~-47°
- dipoles exist (positive at N terminus)
- some amino acid residues are helix breakers (glycine and proline)
24
Q
Features of beta structures (8)
A
- peptides with more extended structure than alpha helix
- hydrogen bonding occurs between adjacent chains
- beta sheets are 2 or more beta structures
- ~6 amino acids residues in length and can have up to 15 residues
- sheets can be parallel or antiparallel
- side chains point above and below the sheets
- any NP-P-NP-P normally forms B-strand
- sheets not planar and one is pleated with a right hand twist
25
What are turns and their features
When a secondary structure turns on itself.
- needed to form globules
- often short and hairpin like
- high gly, pro content
- H bonding across bottom to stabilise
- almost 30% of residues are involved in turns
- more than 16 types
26
What are tertiary and quaternary structures?
Tertiary are multiple secondary structures folded together and quaternary are multiple tertiary structures
27
How are protein structures displayed?
Alpha helix as spirals or cylinders, beta structures as arrows pointing N->C and turns/coils as loops or rope-like stretches
28
What are the four types of super secondary structure?
Helix-turn-helix, B-hairpin, Greek Key, Strand-helix-strand
29
What provides the most structural support for a protein
Hydrophobic core
30
What are the protein groups based on tertiary structure?
A-domain family: four helix bundle and globin fold
A/B family: a/B barrel and a/B horseshoe
Anti-parallel B family
31
Who proved that proteins fold spontaneously and how
Christian Anfinsen used urea and B-mercaptoethanol, then removed and watched it refold itself
32
What is the likely protein folding pathway
Formation of short secondary structures-> nuclei/ subdomains forming (super secondary structures)-> subdomains come together to form partially folded domain-> final domain structure appears
33
What are different non-covalent bonds which contribute to protein stability? (6)
Hydrophobic interactions, hydrogen bonds, metal ion coordination, disulfide bond, side chain hydrogen bonding and salt links (anion/cation)
34
What do chaperones do and what is it called when a protein needs extra help
They help proteins fold up
| Chaperonin-dependent= ~15% of proteins need this
35
What can cause protein unfolding? Which are reversible and irreversible?
Change in pH and heating are irreversible
Detergents and organic solvents (unknown)
Urea and guaridium HCL reversible
36
Examples of misfolded proteins leading to disease
- prions: kuru. Causes misfolding of a->B of PrP protein. No treatment and always fatal, causes more proteins to misfold the same
- alzheimers and Type II diabetes are from amyloid protein misfolding
37
What are the functions of haemoglobin and myoglobin
Myoglobin stores oxygen in the muscles and haemoglobin transports oxygen in the blood to tissues
38
What is oxygen availability like in animals
Limited. Muscles burn more oxygen than we can provide to it. Exercising tissue and the brain are limited to availability
39
Structural features of myoglobin
- ~150 amino acids
- secondary structure: 8 alpha helices A-H and connecting loops
- tertiary structure: globin fold with hydrophobic pocket, haem binds to His F8 in globin protein
- quaternary structure: monomeric
40
Structure of haem
- prosthetic group or co-factor
- 4 pyrole rings in a plane (protoporphyrin)
- Fe has 6 bonds at 90°; 4x N from pyrole rings, N from HisF8 and O2
- Fe-O2 is a reversible reaction
- molecular electronic orbitals give the red colour
41
What is spectroscopy and the law used
Light absorbance can tell us the concentration of substances in a mixture. Beer-Lamben Law converts from absorbance to concentration. Different wavelengths are absorbed more or less efficiently
42
How can spectroscopy be used to myoglobin and haemoglobin structure?
On a spectrum, 2 peaks shows O2 is bound and 1 peak means the molecule is O2 free. Haem has visible absorbance differing from bright red HbO2 (oxygen bound) and dull red Hb (oxygen free)
43
How is haem-O2 in myoglobin?
By co-ordinate linkage to HisF8 and HisE7. HisE7 distorts the binding of gas to the 6th co-ordination position on Haem Fe. This reduces the binding affinity of O2, weakening the bond and making it easier to release the O2 molecule when it is needed
44
What is allosteric control?
Describes how affinity for O2 is altered by molecules binding elsewhere and builds on steric hinderance eg lactate in myoglobin
45
Structural features of haemoglobin
- tetramer (4 globin proteins associate together non-covalently)
- each globin protein contains one haem and can each bind to one O2
- has symmetry with 2x 2 halves interacting with one another
46
What are the haemoglobin conformations and what does it mean
Aerobic zone with O2 present, needle-shaped crystals occur
Anaerobic zone without O2 present, hexagonal plate-shaped crystals occur
Means there is a shape change with haemoglobin when O2 binds and unbinds from haem. Any haemoglobin molecule can switch between both structures
47
What does O2 availability to cellular proteins depend on?
- pO2 in the local environment
| - binding affinity of O2 to haemoglobin
48
Shape of myoglobin binding and release of O2 binding and release on graph
Hyperbolic curve due to tight binding to O2 and saturation at low pO2
49
Shape of haemoglobin binding and release of O2 binding and release on graph
Sigmoidal curve from weak binding as there is more O2 available in the lungs and the weak binding allows for easy dumping of the O2 at tissues
50
MWC concerted model for haemoglobin binding
Subunits can be low-activity tense (T) or high-activity relaxed (R). All 4 units are in the same state of either T or R. Binding of each O2 shifts equilibrium more in favour of R. Inhibitors stabilise T form and activators stabilise R form
51
An example of another protein model which isnt used for haemoglobin
KNF sequential model. Substrate binding causes T->R conformational change in one subunit
52
What things do haemoglobin and myoglobin have in common
- O2 binds to Fe of haem
- shift from dull to bright red allows for monitoring O2 binding
- affinity for O2 is altered by molecules binding elsewhere (allosteric control)
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What things do haemoglobin and myoglobin differ in
- monomer v tetramer
- tighter, hyperbolic bonding curve v weaker, sigmoidal bonding curve
- store O2 in tissue v transport molecule
54
What is the shape of haem in deoxyhaemoglobin and oxyhaemoglobin?
Deoxyhaemoglobin: dished shaped haem
Oxyhaemoglobin: flattened haem by O2 binding
55
How does O2 binding flatten haem?
Fe is moved into plane of haem, pulling HisF8 down and repositioning helix F. This causes shifting in orientation of protein’s secondary elements (such as helix F moving relative to helix C) called conformational changes
56
Side chain packing in R- and T- states
Taught T state= deoxyhaemoglobin and relaxed R-state= oxyhaemoglobin.
A-helices move to different stable positions and not much time is spent in an intermediate state
57
What opens during the T-state
Distance in the middle leading to a binding site for 2,3-biosphosphoglycerate (BPG)
58
What is BPG?
Allosteric inhibitor of O2, negatively charged causing electrostatic interactions allowing BPG to bind to deoxy-Hb.
Stabilises Hb in deoxy T-state due to reduction in O2 affinity
59
When is BPG produced and where
Produced during respiration in peripheral tissues so promotes O2 release where it is needed
60
What promotes cooperativity and what things can do this
Allosteric inhibitors. BPG, Co2 (binds to extreme N terminals amino group) and reduction in pH/incr in H+ (protonate amino acid side chains)
61
How does O2 binding differ in a foetus?
Different amino acid sequences of normal haemoglobin subunits alter O2 binding properties and therefore produce a different haemoglobin. Has different isoforms, especially the gamma isoform with a higher O2 affinity so can get O2 through placenta.
Lacks amino acid in BPG binding site so less able to bind BPG
62
How does binding get affected at higher altitudes?
Higher altitude leads to increase in BPG from an adaptation to high altitude, causing a decrease in haemoglobin O2 binding and more O2 to the tissues due to higher affinity
63
What is methaemoglobin and how is it created
Damaged haemoglobin from oxidation of haem Fe2-> Fe3 shifting one subunit to the R-state conformation without O2 bound.
64
2 ways which methaemoglobin causes impairment
- subunit doesnt bind O2 despite otherwise being in the R state
- other subunits of tetramers are shifted to the R-state so they don’t release O2 in tissues as they should
65
What fixes methaemoglobin back to haemoglobin?
Enzyme cytochrome b5 reductase reduces methaemoglobin using transfer of electrons from NADH
66
What is another type of damaged haemoglobin?
HbM- boston haemoglobin is an example of it, HisE7 -> TyrE7 changes the environment leading to oxidation. Haem plane moves slightly breaking the Fe-HisF8 connection. Remains in T-state with low affinity for O2
67
How is sickle cell haemoglobin caused (HbS)
Gain of function mutation where haemoglobin polymerisation occurs. B E6V mutation causing polymerisation of tetramers to stack up into a chain and distorts cells by making their ends poke out.
68
Effects of HbS in person
Sickle cell, blood cells are deformed and can get stuck in blood capillaries and less able to transport O2
69
What is an enzyme
Biological catalysts which increase the rate of reaction. Most are proteins
70
What is cellular integrity
Decrease in entropy in the cell so energy from elsewhere is required. Enzymes control where and when energy is released to maintain the cell
71
Enzymes in relation to speed throughout reaction, what do they do to achieve a faster reaction?
Reactions pass through high energy transition states which require activation energy to reach. Enzymes lower this activation energy required to get to the transition state and dont interfere with the gibbs free energy of the reaction
72
Examples that show no relationship between enzymes and gibbs free energy
Aldolase- positive G, big rate enhancement
| Adenylate kinase- near 0 G, big rate enhancement
73
Enzyme nomenclature
Enzyme Commission set up to design standardised method for naming enzymes. Over 7000 different enzyme catalysed reactions have been characterised.
74
What is an isozyme
Enzymes which differ in sequence but catalyse the same reaction
75
6 classes of enzymes
1. Oxidoreductase (redox and e transfer)
2. Transferases (transfer functional groups)
3. Hydrolases (hydrolysis reactions with H2O)
4. Lyases (non-hydrolytic bond making and breaking)
5. Isomerases (atom/ group transfer within a molecule to yield isomeric form)
6. Ligases (join 2 molecules together)
76
How does enzyme-substate binding occur
At the active site which has amino acid side chains projecting into it, binds substrate via several weak interactions and determines the specificity of reactions
77
Types of bonds in enzyme-substrate binding
- ionic bonds (aka salt bridges)
- hydrogen bonds (N and O)
- van der Waals interactions (protein and substrate in close proximity. Weak but abundant)
- covalent bonds (strong and rare)
78
Enzyme-substrate models
- lock and key- shapes complementary to each other
| - induced fit- undergoes conformational change and becomes complementary
79
How do enzymes get specificity and reversibility?
Molecular complementarity; many weak interactions. Several bonds required for binding and specificity while weak bonds allow for reversibility.
Weak bonds can only bond if atoms are precisely positioned.
Optimal binding isn’t too tight
80
How is activation energy lowered?
1. Ground state destabilisation
2. Transition state stabilisation
3. Alternative reaction pathway with a different lower energy transition state
Both 1 and 2 achieved by having an active site that is shape/ change complementary to the transition state, not substrate
81
What are coenzymes and what do they do
Small organic molecules, co-substrates, carriers of electrons, atoms or functional groups and often derived from vitamins. They help enzymes by sharing electrons
82
Examples of coenzymes
TPP, CoA, FAD and NAD+
83
What are the strategies for catalysis
```
Acid-base catalysis
Covalent catalysis
Redox and radical catalysis (metal ions)
Geometric effects (proximity and orientation)
Stabilisation of transition states
Cofactors with activated groups
```
84
What is covalent catalysis
Formation of a reactive, short-lived intermediate which is covalently attached to the enzyme. Hydrolysis is usually used to remove the connection to the enzyme.
Nucleophilic attach on an electrophile drives covalent catalysis
85
What is acid-base catalysis
Involves ionisable groups and protein transfer. Groups need to be in their correct ionisation state for a catalytic mechanism to proceed. Ionisation may be part of the transition state
86
What does hexokinase do
Uses Mg2+ as a cofactor, establishes orientation of phosphates of ATP by octahedral coordination of Mg ions.
The electron withdrawing lewis acid stabilised electrons on oxygen making phosphorus a better electrophile
87
How do enzymes relate to pH
Enzyme activity is pH dependent. Each enzyme has a characteristic optimal pH at which its rate is highest- needs to be protonated or deprotonated depending on its pKa
88
How does histidine work
Involved in acid-base hydrolysis with ~6.5 pKa (close to physiological pH so can easily donate or accept protons) depending on the environment of the active site
89
How does chymotrypsin work
Exmaple of an acid-base and covalent catalysis. Works in digestion and is a protease. Chops up/ cleaves proteins. Involves serine, histidine and aspartic acid
90
What allows a triad to work?
Convergent and divergent evolution of enzymes (serine proteases)
91
What is convergent evolution of serine proteases
Some catalytic triads can occur in different order and have different structures. There is no shared ancestor so not in the same family but do the same job
92
What is divergent evolution of serine proteases
Proteins with the same structure do to a common ancestor do similar jobs with unique specifications. Serine proteases recognise different amino acids in scissile bonds
93
What is a progress curve
Measures the appearance of product or disappearance of substrate with time at a steady state
94
How do enzyme concentration and rate of reaction relate
They are proportional
95
How does rate of reaction (velocity) increase with substrate concentration
Velocity increases in a linear way initially but is limited on how fast it can go and reaches a maximum velocity
96
What does the shape of a substrate, velocity curve demonstrate
Shows enzyme properties
97
What determines how well a reaction moves in the forward direction
How easy it is for an enzyme to find/ recognise its substrate
98
What is michaelis constant and what does it mean
KM- substrate concentration at which V(obs)= Vmax/2.
Smaller KM means enzyme is better at finding its substrate
Larger KM meand enzyme is worse at finding its substrate
99
What is the michaelis-menten equation
Substrate, velocity curve described by equation V(obs)=Vmax[S]/KM+[S]
100
What is the michaelis-menten equation used for
Determining kinetic parameters of enzymes
101
What are the assumptions made for the michaelis-menten equation to apply to an enzyme
- product isnt converted back into substrate= irreversible
- Haldane’s steady state assumptions: rate of ES formation= rate of its breakdown
- measuring initial rate ensures [S] doesnt change significantly and [S]>[E]
102
When the michaelis-menten model fits the enzyme rate graph, what is the graph shape and what assumptions are made about the enzyme reaction rate
Hyperbolic shape.
Assumptions: ass ES complexes have same ROR, [S] is in vast excess to [E], Haldane’s steady state assumption, initial rate is measured early enough that [S] doesn’t change significantly and the reverse reaction doesnt occur
103
What is Haldane’s steady state assumption?
Rate of ES formation= rate of it’s breakdown
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When does an enzyme violate michaelis-menten assumptions
When a cell controls enzymatic activity
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How can a cell control enzymatic activity
Enzyme amount, allosteric control, cell location, proteolytic activation, post-translational modification (such as serine phosphorylation)
106
What do allosteric enzymes do and how does this happen
They control metabolic pathways. Respond to effectors binding away from the active site, leading to change in shape and therefore, enzyme activity. They often have multiple subunits and display cooperativity
107
What do cooperativity and allostery have in common
Both depend on enzyme switching between active and inactive forms
108
Examples of allosteric enzymes
Aspartate transcarbamylase (ACTase) and phosphofructokinase
109
What does phosphofructonase control
Glycolysis
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How does cooperativity work in phosphofructokinase
When ATP is high, it is in its inactive T-State as glycolysis isnt needed for energy
When ATP is low, it is in its active R-State as glycolysis is needed for energy
111
What are phosphofructokinase conformations stabilised by
T-state- PEP
| R-state- F6P and ADP
112
How do pancreatic digestive enzymes work
Zymogens are secreted from the pancreas in the inactive form, cleavage by proteases in the gut produce active enzymes and it has temporal and spatial control
113
What is plotted on the lineweaver-burk plot and how are Vmax and Km found
Inverse of Vmax (y) and inverse of [S] (x).
Vmax= reciprocal of y intercept
Km= reciprocal of x intercept
114
What does Km tell us/ its interpretation
Characterises an enzyme-substrate pair. Km= (k-1)/k1
Low Km= high affinity between E and S
High Km= low affinity between E and S
115
What is the physiological significance of Km
In cells, [S] is often below Km for a particular enzyme-substrate interaction, meaning that the rate will rise to accomodate more substrate and tends to maintain a steady state
116
Isozymes and Km and example
Isozymes tend to have different Km due to their different amino acid sequences eg hexokinase for glucose and glucokinase for glucose
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What does turnover number k(cat) mean
Number of S converted into P, per enzyme, per unit of time, when E is saturated with substrate and therefore measures catalytic activity
118
How does Vmax relate to k(cat)
Vmax= k(cat)[E](total)
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What does most effective enzymes have in relation to k(cat) and Km
```
High k(cat) meaning it has ability to turnover a lot of substrate into product per second
Low Km meaning it has a low substrate concentration required to reach Vmax
```
120
What is the measure of overall enzyme efficiency and what does it mean
K(cat)/Km. Higher the better
121
What is the limit for k(cat)/Km and what does it mean
It is the diffusion controlled limit (rate enzyme and substrate diffuse together). Water viscosity sets limit to ~10^9 s-1M-1. Enzymes with k(cat)/Km above 10^8 are referred to as ‘perfect catalysts’
122
What are enzyme inhibitors and why are they important
Compound that binds to an enzyme and reduces its activity. Important because natural inhibitors regulate metabolism and can be used to study enzyme mechanisms and metabolic pathways
123
What are the classes of inhibitors
Irreversible and reversible (competitive and non-competitive)
124
What do irreversible enzyme inhibitors do
Binds to enzyme and permanently inactivates it by reacting with a specific amino acid side chain forming a covalent bond. They often react with catalytic residues and block substrate binding by disabling and filling the active site
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What do reversible enzyme inhibitors do
Bind to an enzyme but can subsequently be released leaving the enzyme in its original condition
126
What do competitive reversible enzyme inhibitors do and what does it cause in relation to Vmax and Km
Inhibitor competes with the substrate for binding on the active site. No change in Vmax and increased Km as more substrate is needed to reach Vmax
127
What do non-competitive reversible enzyme inhibitors do and what does it cause in relation to Vmax and Km
Inhibitor binds to a different site than the substrate meaning its an allosteric inhibitor. The enzyme can bind substrate or inhibitor or both and ES may either not work at all or is just slowed down. Vmax decreases and Km stays the same
128
What is a pure non-competitive inhibitor
Where it has no effect on the binding of the substrate
129
What are transition state analogues, what are they used for and what do they do
Transition state analogues can make ideal enzyme inhibitors as enzymes are often targets for drugs. They make tight binding inhibitors which use a tetrahedral intermediate and inhibit the enzyme (eg adenosine deaminase). Inhibitors can stop drugs from working
130
2 ways which protein activation can occur through
Enzymes and receptors
131
What is a receptor
Cellular protein that controls chemical signalling between and within cells. Targets of drugs and toxins
132
Enzymes v receptors binding sites
Enzyme; one active site
| Receptor; can have several binding sites
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Enzymes v receptors what they bind
Enzymes; substrates
| Receptors; ligands
134
Enzymes v receptors how they work
Enzymes; substrate to products
| Receptors; release the ligand unchanged once the job is done
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Enzymes v receptors where they’re found
Both membrane bound or free
136
Something enzymes and receptors have in common
Both can be activated and inhibited and used as drug targets
137
Different receptor classes
Ligand-gated ion channels
G protein-coupled receptor (GPCR)
Receptor tyrosine kinase (RTK)/ enzyme-linked/ kinase-linked receptor
138
Steps for protein activation and inhibition
Chemical substances travels from its source
Binding to receptor proteins
Causes activation on inhibition
Changes cellular response/ causes it
139
What is a chemical receptor which binds to a receptor
Iigand
140
What are features of lignads
Diverse in structure, endogenous produced in body, exogenous are drugs and toxins, all make chemical contacts with specific receptors
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What must be present for ligand-receptor binding to work
Specificity
142
How do drugs use specificity to work
By tweaking an endogenous ligand, an effective medicine can be used to bind to receptors using the endogenous ligand specificity
143
What type of ligand causes activation and what type causes inhibition
```
Activation= agonist
Inhibition= antagonist
```
144
What happens to cause a cellular response (what is it called)
Signal transduction
145
what is signal transduction
When an active receptor starts a chain of events where messages are passed on through the cell. Often multi-step pathways provide opportunities for coordination and regulation of the chemical response
146
What are second messengers
Transmit signals from a receptor to other relay molecules because they aren’t attached to the membrane and are free to move in the cell. Used by many different receptors
147
What are GPCRs and the most common types
G-protein coupled receptors which use G-proteins to start signal transductions
Gas- stimulatory G-protein which activates enzyme adenylate cyclase
Gai- inhibitory G-protein which decreases the activity of adenylate cyclase
148
What are RTKs
Receptor tyrosine-kinase which used phosphorylation of adaptor proteins to start signal transduction
149
How does phosphorylation work
When protein kinases transfer phosphate from ATP to protein
150
How does dephosphorylation work
When protein phosphatases rapidly remove phosphates from proteins to control signal transduction
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How does ligand-gated ion channel signal transduction work
Agonist ligand binds to receptor and causes change to activate it. This leads to ions directly flowing through the channel into the cell to produce effects
152
How do different responses work
Responses can be controlled by where receptors are expressed. Different cells have different collections of receptors and relay molecules allowing cells to detect and respond differently to different ligands as different combinations can be used such as pathway branching and crosstalk
153
How does insulin work in muscle and adipose tissue
Receptor activation causes phosphorylation of adaptor protein and further signal transduction events cause GLUT-4 translocation allowing glucose entry
154
How does insulin work in liver cells
Receptor activation causes phosphorylation of adaptor protein and further signal transduction events cause glucagon synthesis
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How does glucagon work
Receptor activation causes G-protein activation and further signal transduction events leading to glycogen breakdown in liver cells
156
What are insulin, glucagon and GLP-1 examples of
Peptide ligands
157
How does GLP-1 receptor signal transduction work
GLP-1 is produced and released from the gut and acts on pancreatic B cells. The receptor activation causes G-protein activation and further signal transduction events leading to insulin secretion
158
2 main ways which drugs are discovered
- compound -> physiological effect -> molecular target
| - molecular target-> compound -> physiological effect
