GENERAL FEATURES OF ACTIVE SITES

what are some general features of active sites?

1. 3-d cleft or crevice formed by groups that come from different parts of the aa sequence

2. relatively small portion of the total volume of the enzyme

3. unique microenvironment (ex: water is often excluded unless reactant)

4. substrates bind by multiple weak attractions that are reversible

5. specificity of binding depends on the precisely define arrangement of atoms in an active site; they need complementary between active site and substrate

why is less water around the active site is good?

creates a specific environment that promotes activity such as changing the dielectric constant

why is the enzyme so large when the active site is so small?**

enzymes are large due to structural support, helps with folding, stability, and interaction with other molecules. The active site only need to be large enough to bind to the substrate and is highly specific

how complementary are active sites?

1. lock and key: preformed ligand-binding sites that are complementary in shape to their ligand (poor view of binding) → positioning or binding area / catalytic area

2. induced fit: protein does not have optimal complementarity at the binding site in the absence of ligand. Ligand induces a conformational change at the binding site that results in the complementary interaction.

what are some ways to probe an active site?

1. substrate analogs (km)

2. competitive inhibitors (ki)

3. covalent modification agent (irreversible inhibition)

   1. simple: aa R group specific 2. affinity labels (Trojan horse)

4. site directed mutatgenesis

how can we use covalent modification to modify can active site?

1. covalent inactivation: treat enzymes with an agent specific for a particular aa

• ex: DFP , nucleophilic attack

assess change in activity and then track down location of modification with peptide maps or mass spec

2. affinity labeling (trojan horse): substrate analog bearing a covalent modification agent to specifically guide the modification agent to the active site

   • ex: TPCK for chymotrypsin and TLCK for trypsin

     • forms a covalent link to the Phe group taking them to the active site