PDBsum entry: 1kbk

Hydrolase

PDB-id

1kbk

Contents

Description

Header details

Protein chain

Ligands

NAG

Metal ions

_CL

_CA

Waters ×138

* Residue conservation analysis

Tools

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Clefts Calculation

PDB id:

1kbk

Name:

Hydrolase

Title:

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

Structure:

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

Source:

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

UniProt:

P04746 (AMYP_HUMAN)

Seq:

Struc:

Seq:

511 a.a.

Struc:

496 a.a.*

Key:		PfamA domain
		Secondary structure			CATH domain

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

Enzyme class:

E.C.3.2.1.1 [IntEnz] [ExPASy] [KEGG] [BRENDA]

Reaction:

Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.

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

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Key reference

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.

Literature references that cite this PDB file's key reference

PubMed id Reference

17687508 B.Cobucci-Ponzano, F.Conte, M.Rossi, and M.Moracci (2008).
The alpha-L: -fucosidase from Sulfolobus solfataricus.

Extremophiles, 12, 61-68.

18214874 C.A.Tarling, K.Woods, R.Zhang, H.C.Brastianos, G.D.Brayer, R.J.Andersen, and S.G.Withers (2008).
The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts.

Chembiochem, 9, 433-438.

17592362 R.Quezada-Calvillo, C.C.Robayo-Torres, Z.Ao, B.R.Hamaker, A.Quaroni, G.D.Brayer, E.E.Sterchi, S.S.Baker, and B.L.Nichols (2007).
Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.

J Pediatr Gastroenterol Nutr, 45, 32-43.

16511271 S.Z.Fisher, L.Govindasamy, C.Tu, M.Agbandje-McKenna, D.N.Silverman, H.J.Rajaniemi, and R.McKenna (2006).
Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.

Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 88-93.

15722449 R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, and G.D.Brayer (2005).
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.

Protein Sci, 14, 743-755.

PDB codes: 1xgz 1xh0 1xh1 1xh2

15182367 N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, and L.Kandra (2004).
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

Eur J Biochem, 271, 2517-2529.

PDB codes: 1jxj 1nm9

15304511 S.Numao, I.Damager, C.Li, T.M.Wrodnigg, A.Begum, C.M.Overall, G.D.Brayer, and S.G.Withers (2004).
In situ extension as an approach for identifying novel alpha-amylase inhibitors.

J Biol Chem, 279, 48282-48291.

PDB codes: 1u2y 1u30 1u33

12975375 C.A.Tarling, S.He, G.Sulzenbacher, C.Bignon, Y.Bourne, B.Henrissat, and S.G.Withers (2003).
Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis.

J Biol Chem, 278, 47394-47399.

12930989 J.E.Nielsen, and J.A.McCammon (2003).
Calculating pKa values in enzyme active sites.

Protein Sci, 12, 1894-1901.

12702721 Y.Tatara, B.R.Lee, T.Yoshida, K.Takahashi, and E.Ichishima (2003).
Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis.

J Biol Chem, 278, 25289-25294.

The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.