Enzymes Flashcards
1
Q
method of action of enzymes
A
- Enzymes can couple a spontaneous reaction to a nonspontaneous one, to make the overall ΔG < 0 (spontaneous)
- Reactions pass through high energy transition states.
- Activation energy is required to reach the transition state.
- Enzymes catalyse thermodynamically favourable reactions by lowering the activation energy
- The overall ΔG for the reaction is not changed.
2
Q
what are enzymes made of
A
usually proteins but occasionally RNA
3
Q
6 enzyme classes
A
- oxidoreductase - used for redox
- transferase - transfer of functional group
- isomerases - transfer of atoms/groups withina molecule to form isomer
- lyases - non-hydrolytic breaking or making of bonds
- ligases - join two molecules together
- hydrolase - hydrolysis reactions
4
Q
cofactors
A
non-protein factors which help catalyse reactions. Can be metal ions or coenzymes
5
Q
metal ions as cofactors
A
- Are Lewis acids (i.e. election acceptors), so they can participate in acid-base catalysis
- Form coordination compounds with precise geometries (good for positioning reactants exactly where they need to be).
- e.g. Mg2+ used for DNA polymerase
6
Q
coenzymes
A
- Small organic molecules.
- Co-substrates - required for enzyme-substrate complex interaction, formation or stabilisation
- Carriers (of electrons, atoms or functional groups)
- Often derived from vitamins
7
Q
features of active site
A
- has amino acid side chains pointing into it
- binds substrate via several initial weak interactions
- determines specificity
- initial weak bonds are remodelled to form transition state
8
Q
types of ES bonds
A
- ionic bonds - charged side chains
- hydrogen bonds - O and N atoms in side chains or backbones
- VDW’s interactions - between any protein and substrate in close proximity, weakest
- covalent bonds - rare, v strong
9
Q
why are weak bonds advantageous
A
- easy to break when complex breaks apart - reversibility
- Weak bonds can only form if the relevant atoms are precisely positioned - specificity
10
Q
what does stereospecificity mean?
A
enzymes can recognise between different enantiomers (chiral compounds)
11
Q
lock and key model
A
Substrate and active site have exactly complementary shapes
12
Q
induced fit model
A
- Active site conformation changes slightly when substrate tries to bind
- Shows that enzymes are dynamic, not static
13
Q
3 ways ΔGe‡ is lowered
A
- Ground state destabilisation - free energy increases
- Transition state stabilisation - free energy decreases
- Alternate reaction pathway with a different (lower energy) transition state
(1) and (2) can be achieved the same way: by having an active site that has shape/charge complementarity to the TS, not the substrate
14
Q
should an enzyme bind to substrate or transition state more tightly?
A
transition state however this is difficult because it is transient and cannot be isolated
15
Q
5 catalytic mechanisms
A
- preferential binding of transition state
- proximity and orientation effects - need to be close together and right orientation to react
- acid base catalysis - His is particularly suitable because has pKa 6.5, close to body pH so can donate or accept a proton depending on environment of active site
- metal ion catalysis - provide substrate orientation, ability to act as Lewis acids, sites for electron transfer
- covalent catalysis - formation of a reactive, short-lived intermediate, which is covalently attached to the enzyme
16
Q
progress curve
A
- measures the appearance of product (or disappearance of substrate) with time
- Important to measure initial reaction velocity (rate) i.e. at time zero
- passes through origin
17
Q
factors affecting reaction rate
A
- temperature - increases until optimum
- pH - optimum
- amount of enzyme is increased, the rate of reaction increases, provided substrate is in excess
- As amount of substrate increases, rate of reaction increases linearly until all active sites are occupied, at which point rate stops increasing
18
Q
Vmax
A
- maximum velocity possible when [S] = infinity
- on V vs [S] curve, this is horizontal asymptote
- on Lineweaver-Burk plot, this is 1/y-int
19
Q
what is Km
A
- Michaelis constant
- substrate conc at which V = Vmax/2
- on Lineweaver-Burk plot, Km = -1/x-int
20
Q
significance of Km
A
- Substrate conc needed to reach half Vmax
- Low KM = high affinity between E and S
- High KM = low affinity
- In the cell, for a particular enzyme-substrate interaction, [S] is often below the Km, allowing for rate control
21
Q
kcat
A
- number of substrate molecules converted to product, per enzyme, per unit of time, when E is saturated with substrate
- If Michaelis-Menten model fits then Vmax = kcat[Etotal]
- high kcat is good
22
Q
michaelis-menten equation and assumptions
A
assumptions:
- Product is not converted back to substrate.
- the rate of ES formation equals the rate of its breakdown hence no change in [ES]
- Measuring initial rate means [S] does not change significantly (and [S] is greater than [E])
23
Q
kcat/Km
A
overall measure of enzyme efficiency
24
Q
irreversible inhibitor
A
- Binds covalently to enzyme, permanently inactivating it
- Inhibitor reacts with a specific amino acid side chain, usually in the active site, and forms a covalent bond
- e.g. natural toxins
25
Q
reversible inhibitor
A
- not covalently bound to enzyme
- can be competitive or non-competitive
26
Q
competitive inhibition
A
- Depends on relative concentrations of substrate and inhibitor
- Competes directly with substrate for active site
- No change in Vmax - Infinite [S] outcompetes the inhibitor
- Increases KM - More substrate is needed to get to V = Vmax / 2
27
Q
non-competitive inhibitor
A
- Inhibitor binds does not bind to active site
- Enzyme can bind substrate, or inhibitor, or both
- can be pure non-CI or mixed non-CI
28
Q
pure non-CI inhibitor
A
- Binding of Inhibitor has no effect on the binding of S; i.e. the substrate binds to E and EI with the same affinity
- Binding Inhibitor changes the structure of the active site such that S still binds, but transition state stabilisation is no longer optimal.
- Vmax decreases; KM stays the same
29
Q
mixed non-CI inhibitor
A
- More commonly, binding of the inhibitor does affect binding of the substrate
- Vmax decreases; KM increases
30
Q
methods of enzyme regulation
A
- Turn gene expression on or off
- Degrade enzyme
- Covalent modification e.g. phosphorylation
- Proteolytic cleavage
- Allosteric effects in multisubunit enzymes
31
Q
regulation by proteolysis
A
- Some enzymes are synthesized as zymogens.
- The full length polypeptide is inactive
- Proteases cleave the zymogen polypeptide and remove a peptide or peptides to activate the enzyme
- Many digestive proteases (e.g. trypsin, chymotrypsin) and blood clotting proteins are made as zymogens
32
Q
feedback inhibition
A
- turning an enzyme on AND off
- A sensible strategy is to avoid making unnecessary metabolic intermediates
- Final product blocks an early reaction in a series and shuts down whole series
33
Q
allosteric effect
A
- V vs. [S] plot is a sigmoid, not a hyperbola (like Hb O2 binding curve) (shows Michaelis-Menten curve)
- Allosteric enzymes have multiple subunits
- They display cooperative behaviour:
- Binding of one substrate to the first subunit makes it easier for the second substrate to bind, which makes it easier for the third substrate to bind
- allosteric activators and inhibitors bind to site other than active site
- activator shifts curve left
- inhibitor shifts curve right
34
Q
lineweaver-burke plot
A
- reciprocal of michaelis-menten equation
- x-int = -1/Km
- y-int = 1/Vmax
- slope = Km/Vmax
35
Q
effect of heat on a protein
A
- irreversibly break non covalent bonds (H-bonds) and reform them with other proteins (primarily hydrophobic) – see precipitation with it being unable to dissolve.
- Breaks all the way down to 2o structure
36
Q
effect of salt on protein
A
- removes water from around it, freeing up interactions to other proteins – no real change to 2o structure, and minimal to tertiary (H-bonds mostly the same also).
- Become more compact and shrink so is More stable.
- Can be redissolved
37
Q
what is tyrosinase and what can bind to its active site
A
it is a copper-containing oxidoreductase. Tyrosine, dopamine and O2 can bind to its active site
38
Q
regulation of enzymes by proteolysis
A
- some enzymes synthesised as zymogens (inactive form)
- proteases cleave zymogen to activate enzymes
- allows for temporal and spacial control
