Title : Probing the role of the chloride ion in the mechanism of human
pancreatic alpha-amylase
Abstract :
- Human pancreatic alpha-amylase ( HPA ) is a member of the alpha-amylase family involved in the degradation of starch
- Some members of this family, including HPA , require chloride for maximal activity
- To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced
- Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes
- X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes
- However, the introduction of these mutations did alter the kinetic properties of the enzyme
- Mutations to residue R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA
- In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity
- We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue