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Enzyme Kinetics final- Inhibition and Assays Flashcards | StudBud
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Enzyme Kinetics final- Inhibition and Assays
Enzyme Kinetics final- Inhibition and Assays
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Answers
1
What is incorrect about FRET?
Uniquely suited for assays of lyases and hydrolases
Energy transfer occurs when the donor is close to acceptor (<100 Å).
FRET is uniquely suitable for monitoring degradation reactions
Fluorescence quenching due to the far distance between donor and acceptor
physical phenomenon of energy transfer of a donor and aceptor fluorophores
2
The affinity of kinase for ATP is characterized by _____ and that for inhibitor by_________
1) Ki – dissociation constant of kinase-inhibitor complex
2) KD – dissociation constant of kinase-ATP complex
2) Ki – dissociation constant of kinase-inhibitor complex
1) KD – dissociation constant of kinase-ATP complex
3
What is the mechanism of action of clavulanate?
Inactivates b-lactamase
Hydrolyzes b-lactam residue
Destroys amoxycillin
Prevents bacterial infections
4
Which are Advantages of Mechanism-Based Inactivators
Improved selectivity
Long life-time of the adduct
no danger of random reactions with cellular proteins
Fewer and smaller doses of drug are needed
Inactivate only those enzymes that operate by a certain mechanism
Escape of reactive species cause side effects
5
How is the binding constant obtained in the functional assay?
By studying the initial reaction rates of enzymatic rxn with inhibitors
By monitoring a spectroscopic property
By determining the inhibition mode
By quantitating enzyme product vs. time
6
Enzyme-Ligand Binding Assays – Differential Scanning Calorimetry (DSC)
Is not as Rigorous as the one that involves Isothermar titration
Ligand should not bind to partially unfolded forms
[L] >>> KD
Differential counting calorimetry is the method which can evaluate inhibito
The melting transition is reversible – condition not met in many cases
All the Above
7
What is the function of an antimetabolite?
Activates metabolic pathways
Inhibits metabolite use
Creates new metabolites
Increases enzyme activity
8
Which is correct for Coupled Enzyme Assays?
The first reaction should be rate-limiting, the read-out reaction is fast
inhibition of the first reaction E1 is studied
Used when the enzyme of interest does not produce an easily readable signal
the second reaction catalyzed by E2 produces the signal.
9
IC
50
= concentration of inhibitor that reduces activity of a protein to half-maximal value.
True
False
10
A drug that displaces ATP from its binding site is known as a
Mixed
Uncompetitive Inhibitor
competitive inhibitor
Non competitive inhibitor
11
Enzyme-Ligand Binding Assays – Isothermal Calorimetry (ITC)
When saturated, adding more ligand has no enthalpic effect
The Largest Spokes are seen when all of the ligand is forming the complex
All the Above
assay requires larger amounts of pure protein
12
Have low pKa leaving group that makes them reactive, problem of high background noise for rns with low enzyme activities
True
False
13
What is important for the making of Mechanism-Based Inactivators
Knowledge of side effects of drugs.
Knowledge of enzyme mechanism necessary.
Knowledge of protein signaling.
Knowledge of pharmacology of drug.
14
What is required to determine Ki in enzyme assays?
Using analytical tools
Knowing the inhibition mode
Quantitating enzyme product
Monitoring spectroscopic properties
15
Which is incorrect for
Evaluating Inhibitor Potency – Enzyme Assays
?
You use this assay to derive Km and Vmax from rate of enzymatic rxn
Products of enzymes are not amenable to direct quantification by spectrosco
Methods are the same even if you change substrate
If Vmax is known for enzyme, the assay can be used to quantify the # of it
16
Which is correct for mechanism-Based Enzyme Inactivators
Reactive species seek out a nucleophilic residue in the active site
Chemically unreactive.
Structures are similar to substrate or product.
All the above
ØInactivation occurs prior to release of the activated species.
Target enzyme converts the inhibitor into a reactive that inactivate enzyme
17
How do antimetabolites affect the metabolic pathway?
They block enzyme activity
They get incorporated into it
They attach to the end of the pathway
They prevent metabolite production
18
What do Mechanism Based Inactivators differ from Targeted Covalent Inhibitors?
initially unreactive
target enzyme activates inhibitor by Hydrolysis
target enzyme activates inhibitor by catalysis
19
What is the main reason for resistance to amoxycillin?
Inactivation of b-lactamase
Overproduction of b-lactamase
Hydrolysis of b-lactam residue
Clavulanate inhibitor
20
Clavulanate inactivates b-lactamase by normal catalytic step of this enzyme following formation of the reactive interm
False
True
21
Similarly to TCI, inhibition by mechanism-based inactivators is
not
slowed by a substrate or a competitive inhibitor
True
False
22
Which is incorrect for Determining Binding Constants – Binding Assay? BD = Binding Constant
Fractions of bound protein is plotted as a function of ligand concentration
Kd is obtained directly from the chemical rxn
Inflection point on the semi-log binding isotherm is Kd (50% bound prot)
BC based on distinct properties of free protein vs. protein-ligand complex.
23
How many antimetabolite drugs are approved for cancer treatment?
5
7
21
14
