Handout 11 - General Aspects of Active Sites

What are the features of active sites?

1. 3D cleft/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 (I.e - water is often excluded unless it is a reactant, influences electrostatic intermediate; microenvironment optimal for job that needs to be done)

4. Substrates bind by multiple weak attractions that are reversible

5. Specificity of binding depends on the precisely defined arrangement of atoms in an active site; needs good complementarity between active sites and substrate (TS)

What are the two different views/general features of active sites?

1. Lock and Key

2. Induced fit

What is Lock and Key?

Pre-formed ligand-binding sites that are complementary in shape to their ligand (poor view of binding; simplistic model)

• 2 parts

• Positioning or binding area (complementary to the substrate)

• Catalytic area (R-groups + prosthetic groups interact with substrate to catalyze reaction)

What is Induced fit?

• Protein does not have optimal complementary at the binding site in the absence of ligand/substrate

• Ligand induces a conformational change at the binding site that results in the complementary interaction

What are the 4 ways to probe an active site (gain info on chemistry of active site)?

1. Substrate analogs (look at km)

2. Competitive Inhibitors (look at ki)

3. Covalent Modification Agent (irreversible inhibition)

4. Site directed mutagenesis

What are substrate analogs?

• Look at km

• Measuring enzyme activity in the presence of different substrates

• Looking to see if small change in substrate influences km

• Km goes up = structural difference made substrate bind more weakly

• Km goes down = structural difference made substrate bind better

What are competitive inhibitors (in regards to active site probing)?

• Look at ki

• Compete with substrate for binding at the active site; but the inhibitors are not chemically changed upon binding.

• Ki = disassociation constant for EI complex

• Smaller ki for given inhibitor = stronger binding/better complementarity

• Bigger ki = weaker binding/worse complementarity

What are covalent modification agents (irreversible inhibition)?

• Simple tools that react with specific R-groups

  • some specific

  • some react with small subset of available R-groups from aa’s

• Simple: aa R-group specific

• More Specific: Affinity Labels (Trojan Horses)

  • more specific to active site of a given enzyme

  • tricks enzymes to binding to active site

• Swoops in for kill by covalently modifying the active site to kill activity

What is site directed mutagenesis?

• Create mutant forms of the enzyme with aa swaps at key positions

  • evaluate performance of mutant relative to control wild type enzyme

Do each of these experiments for active site probing give the whole picture of the active site?

No, just little bits of information.

How is covalent modification used?

1. Covalent inactivation

   • treat enzyme with an agent specific for a particular aa

   • assess change in activity

     • decrease in activity = agent reacted with key aa at active site

   • Track down location of modification with peptide maps or mass spec

What is Peptide Mapping?

• Old style method of separating the fragment on some sort of 2D chromatographic system (with different separation basis in each dimension)

  • polarity, charge, etc

• Tells us which specific aa residue (i.e - the kind, position in sequence) was modified.

  • only small information about active site

• Sequencing only the peptide fragments that showed a change in migration.

How is Affinity Labeling (aka Trojan Horse Inhibitors) used?

• Substrate analog (part of structure that looks like substrate) bearing a covalent modification (electrophile) agent to specifically guide the modification agent to the active site.

  • Examples = TPCK for chymotrypsin; TLCK for trypsin

How is the Trojan Horse analogy applied in the concept of affinity labeling?

• Enzyme active site = city of troy

• Parts of inhibitor = trojans take into city as a victory trophy; the horse

• Greek soldiers hidden inside represent the part of the inhibitor that resulted in destruction of city/inhibited enzyme

In the case of chymotrypsin and trypsin, what is the war head?

• wants to react with histidine

• chloromethyl ketone group

• reactive part that reacts with critical active site of histidine residue

What’s the significance of TPCK for chymotrypsin and TLCK for trypsin?

• TPCK doesn’t react with trypsin

• TLCK doesn’t react with chymotrypsin

• Difficult to freely isolate chymotrypsin and trypsin from each other; always stuck with contamination

  • you can purchase trypsin that has been TPCK treated

    • means that activity has been knocked out and leaves trypsin alone (same deal with TLCK + chymotrypsin)