Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase.
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.
Numao
Shin
S
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Maurus
Robert
R
Sidhu
Gary
G
Wang
Yili
Y
Overall
Christopher M
CM
Brayer
Gary D
GD
Withers
Stephen G
SG
D001483:Base Sequence; D001665:Binding Sites; D002712:Chlorides / Q000502:physiology*; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010179:Pancreas / Q000201:enzymology*; D000516:alpha-Amylases / Q000378:metabolism*
D002712:Chlorides; D000516:alpha-Amylases