CHAPTER 7 - CATALYSIS

B:  MECHANISMS OF ENZYME CATALYSIS

BIOCHEMISTRY - DR. JAKUBOWSKI

 004/12/16

We can apply what we learned about catalysis by small molecules to enzyme-catalyzed reactions. To understand the mechanism of an enzyme-catalyzed reaction, we try to alter as many variables, one at a time, and ascertain the effects of the changes on the activity of the enzyme. Kinetic methods can be used to obtain data from which inferences about the mechanism can be made. Obviously, crystal structures of the enzyme in the presence and absence of a competitive inhibitor give abundant information about possible mechanisms.  It is amazing, however, how much information about enzyme mechanism can be gained even if all you have is a blender, a stopwatch, an impure enzyme, and a few substrates and inhibiting reagents. Systematically, the kineticist, medicinal chemist and molecular biologists (i.e. a well trained chemist) can change:

1. the substrate - for example, changing the leaving group or acyl sustituents of a hydrolyzable substrate;
2. the pH or ionic strength - which can give data about general acids/bases in the active site;
3. the enzyme - by chemical modification of specific amino acids, or through site-specific mutagenesis;
4. the solvent - as will be discussed in the next chapter section .

We will explore in detail the mechanisms of three enzymes. For carboxypeptidase, we will study possible mechanisms for the cleavage of C-terminal hydrophobic amino acids from a peptide. For lysozyme, we will study the structural features of the enzyme and substrates along with the mechanism for cleavage of glycosidic links in bacterial peptidoglycan cell walls.  For chymotrypsin, we will study experiments which vary the substrate, pH, and the enzyme and infer from this information about a mechanism consistent with the experimental data.  Kinetic analyses can be used to determine the:

• order of binding/dissociation of substrates and products
• rate constants for individual steps
• and clues to the nature of catalytic groups found in the enzyme.

B1.  Carboxypeptidase

A peptide substrate binds at the active site of the enzyme. X-ray structures of the enzyme with and without a competitive inhibitor show a large conformational change at the active site when inhibitor or substrate is bound. Without inhibitor, several waters occupy the active site. When inhibitor and presumably substrate are bound, the water leaves (which is entropically favored), and Tyr 248 swings around from near the surface of the protein in the absence of a molecule in the active site to interact with the carboxyl group of the bound molecule, a distance of motion equal to about 1/4 the diameter of the protein. This effectively closes off the active site and expels the water. A Zn2+ ion is present at the active site. It is bound by His 69, His 196, Glu 72, and finally a water molecule as the fourth ligand. A hydrophobic pocket which interacts with the phenolic group of the substrate accounts for the specificity of the protein.

In the catalytic mechanism, Zn2+ helps polarize the labile amide bond, while Glu 270, acting as a general base, which along with Zn2+ helps promote dissociation of a proton from the bound water, making it a better nucleophile.  Water attacks the electrophilic carbon of the sessile bond, with Glu 270 acting as a general base catalyst. The tetrahedral intermediate then collapses, expelling the leaving amine group, which picks up a proton from Glu 270, which now acts as a general acid catalyst.  People used to believe that Tyr 248 acted as a general acid, but mutagenesis showed that Tyr 248 can be replaced with Phe 248 without significant effect on the rate of the reaction.

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