BB Lecture 9-11: Enzymes and Enzyme Kinetics I-III Flashcards
What is the equation for V0?
V0 = (Vmax [S] ) / (Km + [S] )
What is the equation for Km’?
Km’ = Km ( 1 + [I] / Ki )
How do you calculate the extent of the change in Vmax when a non-competitive inhibitor is added?
Vmax’ = (Vmax) / [ 1 + ([I] / Ki) ]
What are transition states and how can they be overcome?
barriers to spontaneous reaction activation energy (G)
What affects the equilibrium of a reaction?
delta G of the reaction (not the enzymes)
What does the enzyme affect?
the reaction rate (not the equilibrium)
What is Le Chatelier’s principle?
a change in the concentration of reactant or product will shift the equilibrium of the reactions involved
What are the two models for enzyme-reactant binding?
lock-and-key
induced-fit
Describe the “first order” region of a reaction.
relationship where the initial rate (V0) is directly proportional to [S]
occurs at low substrate concentrations
Describe the “zero order” region of a reaction.
relationship where the initial rate (V0) becomes constant and the rate is independent of [S]
occurs at very high substrate concentrations
Why might one enzyme (enzyme B) have a higher Vmax than another?
- enzyme B may have more than one active site for this substrate
- enzyme B may operate more effectively using a single active site
How can you tell the relative affinity of an enzyme for a substrate using the Michaelis-Menten plot?
reaching a Vmax at a relatively low substrate concentration indicates a higher affinity for substrate
What is Km for a reaction that shows a typical hyperbolic relationship?
the substrate concentration at which V0 is 50% of Vmax
What is the Michaelis-Menten equation?
V0 = ( Vmax [S] ) / ( Km + [S] )
How can the Michaelis-Menten equation be modified for first order behavior?
used if substrate concentration is much lower than Km
V0=[S]
How can the Michaelis-Menten equation be modified for sero order behavior?
used if substrate concentration is much higher than Km
V0=Vmax
What is the best way to determine Vmax and Km accurately?
Lineweave-Burke transformation (double-reciprocal transformation)
What is the Lineweaver-Burk equation?
1/V0 = (Km/Vmax) x (1/[S]) + (1/Vmax)
What is a competitive inhibitor?
compounds that are structurally similar to the substrate of an enzyme but are not the substrate and are not altered by the enzyme
compete for access to the active site
How can a competitive inhibitor be overcome?
increasing substrate concentration
What would a competitive inhibitory do to a Lineweaver-Burk plot?
y-intercept (1/Vmax) will be unaffected
slope (Km/Vmax) becomes steeper resulting in a rightward shift (increase) in the x-intercept (-1/Km)
How can we use the Michaelis-Menten equation to describe the relationship between V0 and [S] in the presence of a competitive inhibitor?
Km’ = Km (1 + [I] / KI)
where:
[I] = concentration of the inhibitor
KI = dissociation constant of the inhibitor for its enzyme binding site
What is the IC50?
measure of an inhibitor’s potency
half of the maximal inhibitory effect
How can the IC50 be found graphically?
when plotted on a graph with [Inhibitor] (M) on the x-axis and % Inhibition on the y-axis draw a dotted line across at the point of no inhibition and at max inhibition and find the middle on the y-axis
How can you determine the IC50 from an inhibitor’s potency?
IC50 = KI (1 + [S]/Km)
* Note when [S] is constant IC50 and KI are directly related
What is a non-competitive inhibitor?
a compound that does not bind at the substrate-binding site (and cannot be displaced by increased [S])
interferes with enzyme’s ability to catalyze the reaction
How does adding a non-competitive inhibitor impact a Lineweaver-Burk plot?
increase the y-intercept (1/Vmax)
x-intercept (-1/Km) is unchanged
slope becomes steeper
How can you calculate the extent of the increase in 1/Vmax for a non-competitive inhibitor?
Vmax’ = Vmax / (1 + [I]/KI)
What is an uncompetitive inhibitor?
binds after the substrate is bound to stabilize the ES complex
slows the dissociation of substrate from enzyme/formation of product
How does adding an uncompetitive inhibitor impact a Lineweaver-Burk plot?
decreases Km
decreases Vmax
normally at the same magnitude
results in upward parallel displacement of the line
What is irreversible inhibition?
inhibitors that form a covalent and essentially irreversible bond with the enzyme
reduces the number of functional enzyme molecules
How does adding an irreversible inhibitor impact a Lineweaver-Burk plot?
decreases Vmax
does not change Km
looks like a non-competitive inhibitor
What unique properties do irreversible inhibitors have?
1) as duration of exposure to the inhibitor increases, so does the number of affected enzymes, so the longer it is present the greater the reduction in Vmax
2. even if the inhibitor is removed the enzymes remain inhibited so enzymatic function can only be recovered when more enzyme is synthesized
What is a modulator/effector?
i. a substrate for the enzyme (in which case the modulatory site is the same as the active site)
ii. some other molecule that acts outside the active site
What are allosteric enzymes and how are they typically regulated?
enzymes that change their conformational enzemble upon the binding of an effector (allosteric modulator)
results in an apparent change in the binding affinity at a binding site
What is homotropic modulation?
when the substrate is the modulator for an enzyme
enzymes regulated by this typically have multiple active sites
What is positive cooperativity?
when affinity at a subsequent active site is increased by occupation of the first active site
How can positive cooperativity in an enzyme be identified graphically?
sigmoidal shape (rather than hyperbolic)
What is negative cooperativity?
binding of the first substrate molecule decreases affinity for catalytic action at other sites (unusual)
What kinds of reactions do not obey Michaelis-Menten kinetics?
reactions that involve homotropic modulators
K0.5 replaces IC50
What are heterotrophic modulators?
modulators that are not substrates of the enzyme
usually do not bind active site
readily reversible
can play a role in homeostatic control
What kinds of enzymes are typically regulated by heterotrophic modulators?
rate-limiting enzymes
modulator normally synthesized many steps down stream
(if this is not the case, then the modulator normally does not help maintain homeostatic control)
How do positive modulators (activators) work?
decrease Km without affecting Vmax
How do negative modulators work?
produce an apparently competitive inhibitor (increases Km and no change to Vmax)
OR
apparently non-competitive inhibition (Vmax is reduced Km is unaffected)
What is a zymogen (proenzyme)?
an inactive precursor of an enzyme that contains the sequence of the active enzyme and must be cleaved from the enzyme by a previous product in the pathway to become activated
Provide examples of zymogens and why their regulation is important.
trypsinogen and chymotrypsinogen (often end in -ogen)
some enzymes are made in different places than where they are used and their activity would be detrimental to the place they are made, so synthesizing the inactive form and having the enzyme to activate it at the location where it is needed is favorable
it is also important for functions were an inactive form can be stored up for use in acute situations (eg. the clotting pathway)
What are isozymes?
enzymes that perform the same function
reflect the fact that the same reaction may require different kinds of regulations or substrate concentrations in different tissues or cells
What is kcat?
the number of operations that a single molecule of enzyme can perform per second
not intrinsic to the enzyme
dependent on the substrate the enzyme acts on
What is the equation for the turnover number?
kcat = Vmax / [Et]
where [Et] is the molar quantity of enzyme present in a reaction
How do you compare enzyme efficiency under physiological conditions?
specificity constant
because under physiological conditions [S] rarely approaches saturation levels, which is used to calculate kcat
How do you calculate the specificity constant?
specificity constant = kcat/Km
What does the specificity constant indicate?
how efficient the enzyme is when free binding sites are abundant
What is an apoenzyme?
the protein component of a holoenzyme that is covalently bound to a cofactor
What is a holoenzyme?
the complex of an enzyme and a cofactor
Are all enzymes proteins?
No- ribosomes are enzymes composed of RNA
