UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Mechanistic studies on human pancreatic [alpha]-amylase Rydberg, Edwin Harold

Abstract

Human pancreatic α-amylase (HPA) is known to hydrolyze maltooligosaccharides with retention of anomeric configuration utilizing at least two active site carboxylic acids as nucleophile and acid/base catalyst in a double displacement reaction. The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of HPA were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the recombinant enzymes. All three residues were altered to both alanine and the corresponding amide, in addition several other variants were generated (E233A/D300A, E233K, D300H, D300Y). The ten mutants, and the wild-type HPA, were successfully expressed in Pichia pastoris, and were secreted at modest levels (10 mg/L). Isolation of the mutant proteins required development of a new purification strategy since the affinity precipitation used to purify wild-type HPA was demonstrated to be ineffective on the low activity ocamylases. Hydrophobic interaction chromatography followed by anion exchange chromatography provided the desired mutant enzymes in highly purified form. In addition, the recombinant enzymes were shown, by electrospray ionization mass spectrometry (ESI-MS) in combination with endoglycosidase F treatment, to be heterogeneously glycosylated at a single asparagine (N461). Partial deglycosylation of the recombinant enzymes allowed for their successful crystallization and subsequent structural characterization to 1.8 Å resolution. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, since virtually all activity (>10 ⁵ fold decrease in k[sub cat] at values) was lost for the enzymes mutated at this position when assayed with each substrate. The significantly greater second order rate constant of E233 mutants on "activated" substrates (k[sub cat /K[sub m] value for α-maltotriosyl fluoride = 15 s⁻¹mM⁻¹) compared with "unactivated" substrates (k[sub cat /K[sub m] value for maltopentaose = 0.0030 s⁻¹ mM⁻¹) strongly suggested that E233 is the general acid catalyst. Data for the D300 mutants will be presented, although the role of D300 remains unclear. In conclusion, structural and kinetic analyses of wild-type and mutant HP As have provided strong evidence for the roles of two conserved active site carboxylic acids (D197 and E233) in its double displacement mechanism, while the role of D300 remains elusive.

Item Media

Item Citations and Data

Rights

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics