|Lecture Notes: 22 February
© R. Paselk 2006
Enzyme Catalysis, cont.
Stabilization of Transition State Conformation: (Strain/distortion; Charge neutralization)
Strain/distortion example: Alkyl phosphate hydrolysis:
The base-catalyzed hydrolysis of A takes place more than one-hundred-million times faster than that of B, apparently due to the strain in the five membered ring in A.
Metal ion catalysis: metal ions can act as electrophilic catalysts in covalent type catalytic mechanisms and also as counter ions in charge stabilization in TS catalytic mechanisms.
Enzyme Examples of Catalysis
Now want to put it all together and look at example enzymes to try to explain their activities.
Lysozyme: Have looked at model of Lysozyme - globular with cleft to accommodate substrate (overhead; model). Functions as an antibiotic, hydrolyzing polysaccharide strand in cell walls of bacteria. For Lysozyme the substrate is a carbohydrate polymer. (Figure 6.17) [overhead].
- X-ray diffraction images show many H-bonding sites to substrate; Glu-35 and Asp-52 are in the active site near the bond to be cleaved. [overhead]
- In binding studies find that binding energies increase as go from 1-4 residues, then decrease on 5 and increase again with 6.
- Lysozyme has a symmetrical pH profile similar to papain with a pH optimum of 5, with Glu-35 unionized (pKa= 6.5) and Asp-52 ionized (pKa= 3.5). Note pK's of both of these residues are different than expected in free amino acids. Very typical in proteins due to local environments. Thus for Glu see pK shifted up because of local hydrophobic, non-polar environment (ions unstable in non-polar environment), whereas the Asp has an environment encouraging ionization.
- So now let's look at the enzyme itself:
- Lysozyme uses a variety of catalytic mechanisms: General acid/base, TS strain distortion, & TS charge stabilization. Let's look at mechanism
- Use binding energy to distort to half-chair conformation (C1 goes from sp3 to sp2-like geometry it will have in TS carbocation intermediate (Figure 6.19) [overhead].
- Ionized (-) Asp-52 acts to stabilize (+) carbocation charge in TS. (Figure 6.20) [overheads]
- Protonated Glu-35 acts as general acid (proton donor) to catalyze cleavage of glycosidic bond, then as general base (proton acceptor) to catalyze attack of water on carbocation intermediate. (Figure 6.20) [overheads]
- In addition each of the catalytic groups is placed in close proximity and properly oriented, so we get proximity/orientation catalysis of catalysis.
Chymotrypsin: (digestive enzyme: zymogens-precursor protein has peptide covering active site, activated by having it hydrolyzed off).
- Asp-His-Ser catalytic triad in serine proteases (Figure 6.25). [overhead]
- Mechanism, as noted in Figure 6.27. [overhead]
The carbohydrates, or sugars, are our third group of biomolecules. They are characterized by having a carbonyl carbon (aldehyde or ketone) and multiple hydroxyl groups. The smallest sugars are thus the three carbon trioses, glyceraldehyde (aldotriose) and dihydroxyacetone (ketotriose).
Note that sugars occur in both D and L forms. As we shall see the natural sugars are generally D.
Last modified 22 February 2007