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 and organic chemist can change:
- the substrate - for example, changing the leaving group or acyl sustituents of a hydrolyzable substrate;
- the pH or ionic strength - which can give data about general acids/bases in the active site;
- the enzyme - by chemical modification of specific amino acids, or through site-specific mutagenesis;
- 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.