BCH 4053 Biochemistry IFall 2001
Dr. Michael Blaber
Enzyme Inhibition, Reactions Involving Two or More Substrates, Ribozymes and Abzymes
Inhibitors are molecules or compounds that interact with an enzyme to as to decrease the activity of the enzyme. There are two general categories of inhibitors:
Irreversible inhibitors. Interact via stable, covalent interactions with the enzyme
- Reversible inhibitors. Have affinity for an enzyme via
There are two major categories of reversible inhibitors: competitive reversible inhibitors, and noncompetitive reversible inhibitors:
The inhibitor (I) competes with the substrate (S) for the enzyme active site (also known as the S-binding site). Binding of either of these molecules in the active site is a mutually exclusive event
- The substrate and inhibitor share a high degree of structural similarity. However, the inhibitor cannot proceed through the reaction to produce product.
- Increasing the concentration of substrate will outcompete the inhibitor for binding to the enzyme active site
- A competitive reversible inhibitor can be identified by its characteristic effects upon kinetic data
Inhibitory reaction of enzyme with a competitive inhibitor:
Catalytic reaction of enzyme with substrate:
Briggs/Haldane steady state assumption for [ES] means rate of formation = rate of loss:
Rearrange to solve for [ES]:
Assume a steady state for the [EI] during initial reaction; rate of formation = rate of loss:
Define KI = k-3/k3 (i.e. the enzyme-inhibitor dissociation constant)
The total enzyme concentration equals the sum of any free enzyme plus any in complex with substrate, plus any in complex with inhibitor:
Substitute in the above expressions for [ES] and [EI]:
Solve for [E]:
Recall the relationship between [ES] and Km:
and the relationship between rate of product formation, v, [ES] and k2:
Or, in terms of [E]:
Now, remember that the maximum velocity for product formation occurs if [E]TOTAL = [ES]:
Compare this expression for the reaction velocity in the presence of a reversible competitive inhibitor with the original Michaelis-Menten equation:
The effects of the reversible competitive inhibitor on the kinetics are as follows:
I] = 0) then the equations are the same
As inhibitor is added, the effect is to modify the apparent value of Km. In particular, the apparent Km will be increased by a value equal to (1 + [I]/KI). If Km is increased, the reaction velocity v will decrease.
Note that as [S] gets very large the value of the denominator is essentially equal to [S] and v @ vmax. Thus, the reaction velocity can be driven to vmax with a high enough substrate concentration
- If no inhibitor is present (i.e. if [
The diagnostic criteria for reversible competitive inhibition is that while the apparent Km is affected by addition of the inhibitor, the value of vmax does not change
How is the Lineweaver-Burke double reciprocal plot affected by the presence of a reversible competitive inhibitor?
Noncompetitive inhibitors react with both E and ES (this is because the noncompetitive inhibitor does not bind at the same site in the enzyme as the substrate)
vmax is reduced), but that the affinity for substrate is unaffected (Km remains the same) since the substrate binding site is not occupied by the noncompetitive inhibitor.
- Inhibition cannot be overcome by increasing the concentration of S
- The effect on kinetics is as if the enzyme were less active (
This type of inhibition renders the enzyme "dead" and with no hope of resuscitation.
- A small amount of irreversible inhibitor will look a lot like the reversible noncompetitive inhibitor profile - i.e. reduction in vmax and Km is unaffected.
- The irreversible inhibitor may bind at the active site, or at a different site. The net result is the same - irreversible loss of enzyme activity. However, the "dead" enzyme may still bind substrate if the irreversible inhibitor does not bind at the active site.
- However, reversible inhibitors can be removed by dialysis (i.e. a diffusion process), whereas irreversible inhibitors cannot be removed. Therefore, experiments can be performed to determine whether an inhibitor is irreversible
Kinetics of Enzyme-catalyzed Reactions Involving Two or More Substrates
Enzyme reactions often involve two or more substrates in the reaction:
A + B à
P + Q
A "bisubstrate" reaction
Bisubstrate reactions proceed by one of two possible routes:
- Both A and B are bound to the enzyme, and then the reaction occurs:
E + A + B à
E + P + Q
A "sequential" or "single displacement" bisubstrate reaction
Random single displacement bisubstrate reactions: A or B may bind to the enzyme first (doesn't matter what order they bind)
Ordered single displacement bisubstrate reactions: A must bind first, then B can bind. The 'A' substrate is therefore termed the leading substrate
The A substrate binds first, chemically modifies the enzyme and releases one product. Then, the chemically modified enzyme binds the B substrate and produces the second product (and enzyme is regenerated to original state):
E + A à
E' + P
E' + B à
E + Q
A "ping pong" or "double displacement" bisubstrate reaction
Random single displacement reactions
- All combinations of enzyme, substrate and product are possible:
- The rate-limiting step is the catalytic step: AEB à
- The double reciprocal plot characteristic of random single displacement bisubstrate reactions:
Double displacement bisubstrate reactions ("ping-pong" reactions)
RNA and Antibody Molecules as Enzymes
Catalytic RNA molecules: Ribozymes
It had long been assumed that only proteins could fold into catalytically active structures. However, recently some RNA molecules have been discovered that have catalytic properties. These are called "ribozymes"
Maturation of tRNA precursor molecules by RNAse P enzyme:
Peptide bond formation catalyzed by protein-free 50S ribosomal subunits:
Catalytic antibodies: Abzymes
Antibodies recognize and bind to a specific antigen (a foreign protein or molecule) which elicits the antibody response.
If the antigen is a purposefully-engineered molecule that represents the transition state of a specific enzyme reaction, then the antibody will have a binding site that is complementary to the transition state structure
If an antibody is produced that recognizes the transition state, then if it binds the appropriate substrate it will help to promote formation of the transition state thus, lowering the energy barrier to formation of the transition state, much like an enzyme does
This will increase the rate of catalysis compared to aqueous solutions alone.
© 2001 Dr. Michael Blaber