1BSI

HUMAN PANCREATIC ALPHA-AMYLASE FROM PICHIA PASTORIS, GLYCOSYLATED PROTEIN

1BSI の概要

• エントリーDOI: 10.2210/pdb1bsi/pdb
• 分子名称: ALPHA-AMYLASE, 2-acetamido-2-deoxy-beta-D-glucopyranose, CALCIUM ION, ... (5 entities in total)
• 機能のキーワード: amylase, pichia pastoris, glycosylated protein, mutagenesis, diabetes, catalysis, pancreatic, enzyme, human, hydrolase
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 56228.04

構造登録者

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. (登録日: 1998-08-28, 公開日: 1999-05-18, 最終更新日: 2024-11-20)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2 Å)