Reading & Problems: LNC p. 214-218, p. 210 Fig.6-16; p. 238 prob. 7, Bring a copy of p. 115 of the handout book, or p. 2 of the following handout to class with you!: Triose-P-isomerase illustrations.
A. Reactive groups - Active sites have reactive groups that can catalyze reactions through, base, acid, nucleophile, metal or other mechanisms.
B. High local concentration - The active sites can hold multiple substrates in proximity to each other and in proximity to reactive groups. This high local concentration dramatically increases the likelihood of the reaction between these substrates/groups.
C. Orbital steering - The active site not only holds substrates and reactive groups close together, but actually holds them in an optimal orientation to facilitate a productive interaction.
D. Strain/binding energy - The active site has a shape that binds the transition state most tightly. This can involve bending or stretching of bonds to promote the alterations necessary for the reaction to proceed. The "binding energy" is the energy provided by the formation of the weak interactions between the enzyme and the transition state. The negative binding energy compensates for the positive energy necessary to "bend" the substrate into the higher energy transition state. The enzyme normally will have the highest affinity for the transition state and lower affinity for the substrate and product.
II. Triose phosphate isomerase mechanism as an example.
Another view of the reaction:
Here is a structural view of triose phosphate isomerase Triose phosphate isomerase illustrates how reactive groups, orbital steering, proximity/high local concentration, and strain aid catalysis.
III. Chymotrypsin - hydrolyzes peptide bonds on the carboxyl side of aromatic amino acid residues.
- Protein modification reagents - example diisopropylfluorophosphate (DIFP) reacts with reactive Serine residues.
- Protein affinity labeling reagents - have a structure that is shaped to fit into the active site of the enzyme to target a reactive group to the active site. Example for chymotrypsin: Tosyl-phenylalanine chloromethylketone (TPCK) that reacts with active site His residue.
- Structural determination, especially in the presence of inhibitors/substrate analogs reveals details of active site.
Take home concepts on enzymes:
a) Active site with specific shape and surrounding groups to bind substrate (but usually binds transition state best)
- Catalytic, chemically active groups at active site.
- High local concentration of substrates and active groups
- Orbital steering to maintain reactive orientation
- Stabilization of intermediate forms
- Strain - bending and stretching bonds to aid in formation of transition states
Charles S. Gasser (Department of Molecular & Cellular Biology; UC Davis)