BCH 4053 Biochemistry I
Fall 2001
Dr. Michael Blaber


Lecture 25

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:

  1. Reversible inhibitors. Have affinity for an enzyme via non-covalent interactions
  2. Irreversible inhibitors. Interact via stable, covalent interactions with the enzyme

 

Reversible Inhibition

There are two major categories of reversible inhibitors: competitive reversible inhibitors, and noncompetitive reversible inhibitors:

Competitive 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


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:

therefore:

Or, in terms of [E]:

Thus:

Now, remember that the maximum velocity for product formation occurs if [E]TOTAL = [ES]:

Therefore:


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:

 

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

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)

Irreversible inhibition

This type of inhibition renders the enzyme "dead" and with no hope of resuscitation.

 

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:

  1. Both A and B are bound to the enzyme, and then the reaction occurs:

E + A + B à AEB à PEQ à E + P + Q

A "sequential" or "single displacement" bisubstrate reaction

  1. 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 EA à E'P à E' + P

Then:

E' + B à E'B à EQ à E + Q

A "ping pong" or "double displacement" bisubstrate reaction

 

Random single displacement reactions

Double displacement bisubstrate reactions ("ping-pong" reactions)

 

RNA and Antibody Molecules as Enzymes

Catalytic RNA molecules: Ribozymes

Maturation of tRNA precursor molecules by RNAse P enzyme:

Peptide bond formation catalyzed by protein-free 50S ribosomal subunits:

Catalytic antibodies: Abzymes


© 2001 Dr. Michael Blaber