1BSI

HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN

Summary for 1BSI

• Entry DOI: 10.2210/pdb1bsi/pdb
• Descriptor: ALPHA-AMYLASE, 2-acetamido-2-deoxy-beta-D-glucopyranose, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: amylase, pichia pastoris, glycosylated protein, mutagenesis, diabetes, catalysis, pancreatic, enzyme, human, hydrolase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 56228.04

Authors

Rydberg, E.H.,Sidhu, G.,Vo, H.C.,Hewitt, J.,Cote, H.C.F.,Wang, Y.,Numao, S.,Macgillivray, R.T.A.,Overall, C.M.,Brayer, G.D.,Withers, S.G. (deposition date: 1998-08-28, release date: 1999-05-18, Last modification date: 2024-11-20)

Primary citation

Rydberg, E.H.,Sidhu, G.,Vo, H.C.,Hewitt, J.,Cote, H.C.,Wang, Y.,Numao, S.,MacGillivray, R.T.,Overall, C.M.,Brayer, G.D.,Withers, S.G.
Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.
Protein Sci., 8:635-643, 1999

PubMed Abstract: 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.

PubMed: 10091666

Experimental method

X-RAY DIFFRACTION (2 Å)