PDBsum entry: 1kbk

Hydrolase

PDB id: 1kbk

Contents

Protein chain

496 a.a. *

Ligands

NAG

Metals

_CL

_CA

Waters ×138

* Residue conservation analysis

PDB id:

1kbk

Name:

Hydrolase

Title:

Mechanistic analyses of catalysis in human pancreatic alpha- detailed kinetic and structural studies of mutants of three carboxylic acids

Structure:

Alpha-amylase, pancreatic. Chain: a. Synonym: 1,4-alpha-d-glucan glucanohydrolase, pancreatic al amylase, pa. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: pichia pastoris. Expression_system_taxid: 4922

Resolution:

1.90Å R-factor: 0.171 R-free: 0.177

Authors:

E.H.Rydberg,C.Li,R.Maurus,C.M.Overall,G.D.Brayer,S.G.Withers

Key ref:

E.H.Rydberg et al. (2002). Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids. Biochemistry, 41, 4492-4502. PubMed id: 11914097 DOI: 10.1021/bi011821z

Date:

06-Nov-01 Release date: 10-Apr-02

Protein chain

P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase

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.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (2 terms)
• Biological process: metabolic process (4 terms)
• Biochemical function: catalytic activity (8 terms)

DOI no: 10.1021/bi011821z

Biochemistry 41:4492-4502 (2002)

PubMed id: 11914097

Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.

E.H.Rydberg, C.Li, R.Maurus, C.M.Overall, G.D.Brayer, S.G.Withers.

ABSTRACT

The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.