Tutorial 3 enzymes and enzyme kinetics Flashcards
what are the different ways in which an enzyme can catalyse a reaction?
Acid/base catalysis
covalent catalysis
redox and radical catalysis (metal ions)
geometric effects (proximity and orientation)
stabilisation of transition state
cofactors with activated groups eg electrons, H-, methyl groups (CH3), amino groups (NH2)
Describe the role of each of the side chains and any other features of this active site that enables catalysis
His
- N pulls proton of ser
ser
- O pickup -‘ve charge
- good nucleophile
Asp and His
- share proton between them
- allows his to easily take H+ from ser
Ser
- good nucleophile
- break peptide bond
protease
enzyme breaks down protein
catalytic triad
serine
histidine
asp
oxyanion hole
area +’ve charge from gly193 and ser195
N from substrate attracted to +’ve pocket
specificity
area hydrophobic pocket
correct peptide cut in correct orientation
write true or false for each of the following statements:
a. Enzymes increase rate of reaction
b. Enzymes push the equilibrium of a reaction towards the products
c. Enzymes react with only one or a limited range of substrates
d. Enzymes allow reactions with a +ΔG to have a -ΔG
a. True
b. False
c. True
d. False
where on an enzyme does a substrate bind?
active site
what kinds of interactions bind to a substrate to an enzyme when they first interact?
non covalently (usually)
which part of the amino acids of the enzyme mediates the interactions in above (what kinds of interactions bind to a substrate to an enzyme when they first interact?)?
reactions mediated mostly via sidechains of enzyme that are orientated into the active site
what is the geometric specificity and stereo specificity in the context of enzyme substrate interactions?
enzyme distinguish between stereoisomers *same molecular formula & sequence of bonded atoms, but differ in 3D orientation of their atoms in space.
substrates need to fit into active sites
explain the induced fit hypothesis for enzyme substrate interactions
enzymes will undergo a conformational change when a substrate is bound that will favour the transition state
- lock and key (exact fit)
- induced fit (active site will mould to active site when substrate bind)
in terms of energetics, how do enzymes increase the rate of reaction?
- stabilises transition state
- destabilise the substrate
- provide alternative pathway
how does covalent catalysis differ from non-covalent catalytic reaction mechanisms?
covalent catalysis forms a covalently bound intermediate.
have 2 transition states
(like chymotrypsin enzyme)
what are co-enzymes and what is their role in enzyme catalysed reactions?
co factors
coenzymes help in reaction to function while not part of enzyme itself.
- organic non protein molecules
- derived from vitamins
- carriers in reaction
cofactors
small inorganic ions usually metals. don’t change
coenzymes
- organic non protein molecules
- derived from vitamins
- carriers in reaction
in enzyme kinetics, what is a progress curve?
draw tangent to initial velocity. linear
tangent = Vo
change in absorbance
why does a progress curve become less steep (curve towards flat) with time?
concentration of substrate decreases as the enzyme has converted a lot into products
in enzyme kinetics, what is a V vs [S] curve?
steeper tangent
- increase substrate concentration
- increase reaction rate
use fixed / constant concentration of enzyme
1st part = 1st order kinetics
2nd part = zero order kinetics
- all enzyme active sites occupied / saturates
- reaction can’t go any faster
True or False?
when enzyme assays for a V vs [S] curve are done, enzyme concentration in constant in each assay
True
why does a V vs [S] curve become less step (curve towards flat) with time?
Enzyme becomes saturated with substrate therefore can’t go any faster
Define Km
substrate concentration required to reach half Vmax
Define Vmax
max velocity of an enzyme (very high substrate concentration required)
how does a competitive inhibitor slow down catalysis?
blocks active site for some of the time compete for active site
increase Km - decrease affinity
Vmax unchanged (active site hasn’t changed)
how does a non-competitive inhibitor slow down catalysis?
binds to allosteric site (not active site)
unchange Km in pure.
lower Km if mixed.
Vmax is lowered.
draw a lineweaver-burk plot, including labels for Km and Vmax
margin of error
- further away from points
explain what causes the shape of the curves you drew in 23. below
- draw V vs [S] curve for an allosteric enzyme under negative and positive allosteric effectors
the allosteric binding switches the conformation of the enzyme into either the R or T state, effecting the rate of catalysis by effecting the rate of binding
what is zymogen and why are they useful?
an inactive enzyme that can be activated when it encounters the right conditions
very useful for digestive enzymes, so we don’t digest ourselves
what is a drug
any substance that causes a change in an organisms physiology or psychology when consumed
drugs are typically distinguished from food and substances that provide nutritional support
when a drug enters our bloodstream, our body will do everything it can to detoxify it
which substrate from this table will be preferentially metabolised by alcohol dehydrogenase and why?
methanol Km = 7
ethanol Km = 0.45
Propan-1-ol Km = 0.15
butan-1-ol Km = 0.4
propan-1-ol
low Km affinity of enzymes to substrate
why is antifreeze (ethylene glycol) toxic?
look similar to ethanol
metabolised by ADH
the intermediates are really toxic
as the body tries to detoxify ethylene glycol, it makes more toxic substances, making the toxicity worse.
how is antifreeze poisoning treated?
- administer ethanol
- ADH has a higher affinity for ethanol (0.45 mM) than ethylene glycol (30 mM)
- ethanol will bind to the ADH enzymes instead of ethylene glycol
- ethylene glycol will be detoxified and cleared by alternative pathways
Compare and contrast enzymes and receptors
enzymes
- 1 active site
- binds substrates
- change S into P
- membrane bound or free in cytosol
receptors
- several binding sites
- binds ligands
- release ligand unchanged
- membrane bound or free in cytosol
what receptor does ethanol interact with?
- GABAa receptor
- inhibitory receptor of the CNS
- Agonist activates this receptor, causing Cl- ions to enter into cells
- turns off / dampens down responses
- ethanol agonist of this receptor
what effect does this have at a biochemical level?
ethanol interacts with GABAa receptor
agonist (ethanol) binding opens the channel and allows Cl- into cell
decreases general activity in the brain
effects are dose dependent
- more ethanol more inhibitory effects
what effect does this have at physiological level?
ethanol interacts with GABAa receptor
loss coordination
memory loss
slurred speech etc
low doses
- loss of inhibitions and relaxation
too much
- coma or death
is HIV protease an enzyme or a receptor and why>
enzyme
- active site
- binds substrates and changes it
describe the key features of the HIV protease active site, that help it cleave proteins
aspartate protease
- 2 Asp residues use water to cleave peptide bond
- 2 Asp residues in active site
inhibits enzyme kills virus
not formed in humans
use the lineweaver burk plot to determine if saquinavir acts as a competitive, pure non-competitive or mixed non-competitive inhibitor?
competitor inhibitor
- change in Km
- blocks active site
how could saquinavir be improved?
make close to substrate
make more highly specific to HIV protease to avoid side effects
what can saquinavir cause?
nausea, diarrhoea, vomit
-‘ve effects to drugs you take
