Enzymes Lecture 3 Flashcards
Note: For competitive inhibitions Km stays the same because α = α’ (equation for apparent Km is = α*Km/α’)
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How can we learn more about the way an enzyme
binds a substrate molecule in a reaction?
We can study the inhibitory effect of small molecules (other than the substrate) on the reaction.
______ are important pharmaceutical agents
For example, _____ inhibits the enzyme that catalyzes the first step in the synthesis of prostaglandins (involved in some processes that produce pain)
Enzyme Inhibitors
Aspirin
There are two main classes of inhibitors.
This one is noncovalent binding of the inhibitor; can be reversed by removing the inhibitor
What is one common type?
Reversible Inhibition
Competitive
There are two main classes of inhibitors.
These bind covalently with or destroy an essential functional group on an
enzyme, or form a very stable noncovalent
association
Irreversible Inhibition
This type of inhibitor molecules competes with the substrate molecule for the SAME active site of an enzyme
The inhibitor is very similar to the
Competitive Inhibitor
Substrate
By binding to the enzyme’s active site, the
inhibitor prevents the substrate from binding.
It forms an _____ complex and increases/decreases the rate of formation of ES complex.
Can the inhibitor undergo catalysis?
EI complex
decreases the rate of formation of ES complex
No.
Competitive inhibitors can be analyzed by _____ kinetics
The M-M equation becomes…
At high [S], the ES complex is UNFAVORED/FAVORED so
that the maximum rate can be obtained?
Steady State Kinetics
Vα = Vmax[S]/(αKm + [S])
Favored
What is the equation for α?
What is αKm?
α = 1 + [I]/KI and KI =[E][I]/[EI]
The apparent Km
Figure 11.2 describes the effects of competitive inhibition on enzyme kinetics
____ is the same at high [S]
Lineweaver-Burke plots are _____ as expected for steady state kinetics
Vmax
Linear
Is Vmax changed by the presence of a competitive inhibitor?
No, it stays the same (the y-intercept is the same)
An example of a competitive inhibitor is
shown in Fig 11.3. The molecule UpA is a very good substrate for the enzyme ribonuclease
If the oxygen atom at the cleavage site in the
substrate UpA is replaced by a ____ group to form a phosphonate analog UpcA, a strong competitive inhibitor is formed.
Ribonuclease binds the analog, but it cannot
cleave the _____ bond.
CH2
phosphonate bond
Another example of a competitive inhibitor, this is used in medicine as a treatment for patients who have ingested methanol
It competes effectively with methanol as an alternative substrate for ________
Ethanol
alcohol dehydrogenase
This is another type of reversible inhibitor, it is seen only with enzymes having two or more substrates
It binds at a site different from the substrate active site
Uncompetitive Inhibitor
Unlike a competitive inhibitor, an uncompetitive inhibitor binds only to the ____ complex forming an
Uncompetitive
ES complex
ESI complex
In the M-M equation, in the presence of an uncompetitive inhibitor, the equation is altered to….
Where α’ =
Vα= Vmax[S]/(Km + α’[S])
α’ = 1 + [I]/KI’ and KI’=[ES][I]/[ESI]
Does an uncompetitive inhibitor lower or increase Vmax?
Does the apparent Km increase or decrease? Why?
Lowers Vmax
Decreases because the [S] needed to reach 0.5 Vmax increases by the factor α’
This type of inhibitor binds at a site different from that of the substrate active site, but it binds to either E or ES
Mixed Inhibitor
What is the rate equation describing mixed inhibition?
Vo = Vmax[S]/αKm + α’[S]
How does mixed inhibition affect Km and Vmax?
A decrease in the apparent affinity of the enzyme for the substrate (a decrease in apparent affinity means the Km value appears to increase) or or in an increase in the apparent affinity (an increase in apparent affinity means the Km value appears to decrease) when the inhibitor binds favorably to the enzyme-substrate complex. In either case the inhibition decreases the apparent maximum enzyme reaction rate
When α=α’, it is known as ______ inhibition.
Note this is a special case of mixed inhibition
Noncompetitive
What is the only thing affected during noncompetitive inhibition?
Vmax
What equation can be used to summarize the effects of all reversible inhibitors?
Vo = Vmax[S]/αKm + α’[S]
This category of inhibitors binds covalently with enzymes or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or form a very stable noncovalent association
Irreversible Inhibitors
In one example of irreversible inhibition, ___ reacts with diisopropylfluorophosphate (DIFP) and irreversibly inhibits the enzyme
This has led to the finding that ______ is the key acctive site Ser residue in the above molecule
Chymotrypsin
Ser195
These are a special type of irreversible inhibitor that are unreactive until they bind to the active site of a specific enzyme
Suicide Inactivator
The suicide inactivator undergoes the first
few steps of the reaction, but then it’s
converted into a reactive compound that
combines irreversibly with the enzyme.
They are important in drug design
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Enzymes have an optimal ____ for enzyme activity
Above or below the optimal value, activity decreases
pH
An ionic interaction essential for stabilization of the enzyme’s active conformation might be eliminated.
For example, consider removing a proton from a his residue. The pH range over which an enzyme undergoes changes in activity can provide information as to the type
of amino acid involved
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____ is a digestive enzyme located in the small intestine
Chymotrypsin
Chymotrypsin catalyzes the hydrolytic cleavage of ____ bonds
Peptide
Chymotrypsin is specific for peptide bonds adjacent to _____ residues
Aromatic AA resiudes (Trp, Phe, Tyr)
Chymotrypsin has a widely studied mechanism which includes….
1) General acid-base catalysis
2) Covalent catalysis
3) Transition-state stabilization
Chymotrypsin does not catalyze a direct attack
of water on the peptide bond; a transient
covalent ______ intermediate is formed
acyl enzyme
What are the two phases of the chymotrypsin mechanism?
Acylation, deacylation.
This phase of the chymostrypsin mechanism cleaves peptide bond and form an ester linkage between peptide carbonyl carbon and the enzyme
Acylation
This phase of the chymostrypsin mechanism hydrolyzes the ester linkage and regenerate the nonacylated enzyme
Deacylation
