BICH 441 HONORS, SPRING 2006

Problem Set #2

02/02/06

You isolate an enzyme that splits 4-hydroxy-2-ketovalerate (2-oxo-4-hydroxy pentanoate) into pyruvate and acetaldehyde.
A. Propose a mechanism for this reaction. (hint: see glycolysis)
B. Indicate the nature of functional groups important in your mechanism. Remember to include functional groups required to hold the substrate in the active site as well as groups important for the mechanism.
C. How would you prove the participation of your functional groups? (hint: chemical modification of groups, isotope exchange with solvent, pH rate profiles, etc)

A. aldolase reaction.

B. EITHER metal ion (Zn) polarizes the carbonyl
OR forms a Schiff's base with a Lys
Arg stabilizes carboxyl
Cys or Tyr or His or Asp/Glu base removes proton from 4-OH with aldol cleavage of C3-C4, beta elimination of C4.

C. Metal ion should give a signal using atomic absorption or EPR.
May be able to remove metal ion by dialysis against EDTA or EGTA with inactivation of the enzyme: add back various metal ions to reactivate.
Schiff's base may be reduced with NaBH4 and detected as pyruvoyl or lactoyl adduct of lysine.
Methyl proton of pyruvate will exchange with solvent.
pH rate profile will detect important functional group with pKa of a base.
Specfic reagents for particular amino acids, eg iodoacetate for Cys.