1bsi

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spinqualitylabelsall models 1bsi, resolution 2.Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN

Overview

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic, yeast Pichia pastoris and two mutants (D197A and D197N) of a completely, conserved active site carboxylic acid were generated. All recombinant, proteins were shown by electrospray ionization mass spectrometry (ESI-MS), to be glycosylated and the site of attachment was shown to be Asn461 by, peptide mapping in conjunction with ESI-MS. Treatment of these proteins, with endoglycosidase F demonstrated that they contained a single N-linked, oligosaccharide and yielded a protein product with a single N-acetyl, glucosamine (GlcNAc), which could be crystallized. Solution of the crystal, structure to a resolution of 2.0 A confirmed the location of the glycosyl, group as Asn461 and showed that the recombinant protein had essentially, the same conformation as the native enzyme. The kinetic parameters of the, glycosylated and deglycosylated wild-type proteins were the same while the, k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the, wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm, that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.

About this Structure

1BSI is a Single protein structure of sequence from Homo sapiens with NAG, CA and CL as ligands. Active as Alpha-amylase, with EC number 3.2.1.1 Full crystallographic information is available from OCA.

Reference

Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris., Rydberg EH, Sidhu G, Vo HC, Hewitt J, Cote HC, Wang Y, Numao S, MacGillivray RT, Overall CM, Brayer GD, Withers SG, Protein Sci. 1999 Mar;8(3):635-43. PMID:10091666

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