Enzymes, like all other aspects of living systems, must be controlled.

In general, enzymatic activity can be regulated in two ways:

• Control the amount of an enzyme present by regulating

  • the rate of its synthesis

  • the rate of its degradation

We've already seen methods for switching transcription of DNA on and off, thus regulating the production of proteins.

• Control the enzyme directly:
  • manipulate the enzyme activity

  • manipulate the concentration of its substrate

More rapid methods must also be available.

Irreversible enzyme inhibition is not used within single organisms; it requires covalent binding of an inhibitor that cannot be removed by natural chemical processes.

• Some enzymes can be temporarily inactivated by phosphorylation, but phosphorylated enzymes are readily restored to activity

Nerve gases, such as DFP, are irreversible inhibitors of esterase enzymes, such as acetylcholinesterase, which is involved in nerve transmissions.

The mode of action is shown below.

• Acetylcholinesterase normally functions to hydrolyze the ester group of acetylcholine

• Binding of the quaternary ammonium group in the negative "pocket" positions the ester to react with a serine in another pocket

• DFP reacts with that serine to form a phosphate ester than cannot be removed enzymatically because of the large isopropyl groups

Effective antidotes have been developed that release the active site by binding to the negative pocket, and conducting an internal cleavage of the phosphate. The antidote then can be displace by the enzyme's normal substrate.

Acetylcholinesterase (T. californica; 2ack), Catalytic Residues Emphasized
Catalytic Site of Acetylcholinesterase (1cfj), Reacted with Sarin
Acetylcholinesterase, Quaternary Antidote Bound (2ack)

If irreversible inhibitors form covalent bonds, then reversible ones ought to use weaker modes of bonding, and they do:

• Hydrogen bonds

• Electrostatic interactions (salt bridges)

• Lipophilic interactions

Reversible inhibition typically follows one of four mechanistic schemes:

• Classical competitive inhibition; (a) in the diagram

  The inhibitor competes with the normal substrate for the enzyme's catalytic site.

  If either

  • A large excess of inhibitor is present, or

  • The inhibitor binds more tightly than the substrate

  the enzyme activity will be reduced. Note that after time, if a large excess of substrate builds up, Le Chatelier will restore activity. This is how the statins inhibit HMG-CoA reductase [see Science, 2001, 292, 1160].

• Non-classical competitive inhibition; (b) in the diagram
  • The inhibitor binds to a site other than the active site, producing a sterically inactive, "tense" (= T), state of the enzyme.

  • The normal substrate competes for the free enzyme and binds to the active site to form an active, "relaxed" (= R) state.

  • Obviously, an excess of substrate favors R, and excess inhibitor favors T; tightness of binding can affect the equilibria similarly, as above.

  • This type of inhibition is called allosteric inhibition.

• Uncompetitive inhibition; (c)

  • occurs when the inhibitor binds only to the enzyme-substrate complex, in a site that does not accept inhibitor in the enzyme alone.

  • This kind of inhibition is not reversed by an excess of substrate, since only the complex binds the inhibitor.

• Noncompetitive inhibition; (d)

  • involves inhibitor binding to both the enzyme and the complex of enzyme and substrate, in each case producing a T state.

  • This activity effectively removes enzyme from solution, and cannot be reversed by an excess of substrate.

What does this have to do with regulating enzyme activity? Well, Mother Nature often will use a compound from farther along in the biosynthetic sequence as an inhibitor for an earlier step, usually by allosteric inhibition of type (b). This is called feedback inhibition.