PDBsum entry 3cpu

Hydrolase

PDB id: 3cpu

Contents

Protein chain

496 a.a. *

Ligands

GLC-GLC

Metals

_CL

_CA

Waters ×233

* Residue conservation analysis

PDB id:

3cpu

Name:

Hydrolase

Title:

Subsite mapping of the active site of human pancreatic alpha-amylase using substrates, the pharmacological inhibitor acarbose, and an active site variant

Structure:

Pancreatic alpha-amylase. Chain: a. Synonym: pa,1,4-alpha-d-glucan glucanohydrolase. Engineered: yes. Mutation: yes. Other_details: a part of acarbose inhibitor, disaccharide glc(499)- glc(500) chain b, observed at active site

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: pancreas. Gene: amy2a. Expressed in: pichia pastoris. Expression_system_taxid: 4922

Resolution:

2.00Å R-factor: 0.179

Authors:

G.D.Brayer,G.Sidhu,R.Maurus,E.H.Rydberg,C.Braun,Y.Wang,N.T.Nguyen, C.M.Overall,S.G.Withers

Key ref:

G.D.Brayer et al. (2000). Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry, 39, 4778-4791. PubMed id: 10769135 DOI: 10.1021/bi9921182

Date:

08-Jun-99 Release date: 30-Jun-01

Protein chain

P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase from Homo sapiens

Seq: 511 a.a.

Struc: 496 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.1 - alpha-amylase.
• Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.

DOI no: 10.1021/bi9921182

Biochemistry 39:4778-4791 (2000)

PubMed id: 10769135

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.

G.D.Brayer, G.Sidhu, R.Maurus, E.H.Rydberg, C.Braun, Y.Wang, N.T.Nguyen, C.M.Overall, S.G.Withers.

ABSTRACT

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.