PDBsum entry: 1jxk

Hydrolase

PDB-id: 1jxk

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

491 a.a. *

Metal ions

_CL

_CA

Waters ×323

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

1jxk

Name:

Hydrolase

Title:

Role of ethe mobile loop in the mehanism of human salivary amylase

Structure:

Alpha-amylase, salivary. Chain: a. Fragment: lacking the loop residues 306-310. Synonym: salivary alpha-amyalse. 1,4-alpha-d-glucan glucanohydrolase. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: salivary glands. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108

UniProt:

P04745 (AMY1_HUMAN)

Seq:

Struc:

Seq:

511 a.a.

Struc:

491 a.a.*

Key:	PfamA domain
Secondary structure	CATH domain

* PDB and UniProt seqs differ at 1 residue position (black cross)

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:

0.176

R-free:

0.200

Authors:

N.Ramasubbu,C.Ragunath,Z.Wang

Key ref:

N.Ramasubbu et al. (2003). Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase.. J Mol Biol, 325, 1061-1076. [PubMed id: 12527308] [DOI: 10.1016/S0022-2836(02)01326-8]

Date:

07-Sep-01

Release date:

14-Sep-01

Related entries:

1smd
human salivary amylase: native structure
1jxj
related entry

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1016/S0022-2836(02)01326-8

J Mol Biol 325:1061-1076 (2003)

PubMed id: 12527308

Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase.

N.Ramasubbu, C.Ragunath, P.J.Mishra.

ABSTRACT

Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.

Selected figure(s)

Figure 6.
Figure 6. The overall polypeptide chain fold of the mutant D306:acarbose complex showing the various secondary binding sites of the ligand. Site 2 is anchored around residue Trp284 and is common between the rHSAmy and the D306 structures. This site (site 2) is about 5-6 Å away from the S3' subsite, suggesting a possibility that starch may bind with extended interactions along the surface of the molecule. Site 3 is anchored around Trp383 and is located near the A/C domain interface. An additional maltose residue (site 4) can be seen positioned suitably from the non-reducing end of the active site-bound moiety (site 1). Collectively, these sites give a preliminary view of the mode of starch binding with amylases.

Figure 7.
Figure 7. An illustration of the interactions formed by the ligands bound on the surface of amylase. This Figure was generated using HBPLUS[52.] and LIGPLOT. [53.] (a) Site 2 in the rHSAmy:acarbose complex; only three units were observable in the density maps corresponding to Agl and Glc. The Hmc unit is not seen in the density maps presumably because of disordering. (b) Site 2 in the D306:acarbose complex. Comparison of site 2 of the rHSAmy:acarbose and D306:acarbose complexes shows similarity in the binding site with residues Trp284, Tyr276 providing the stacking interactions. (c) Site 3 in the D306:acarbose complex. Trp383 provides the stacking interaction in site 3 in the mutant structure. In the structure of the D306:acarbose complex, unhydrolyzed acarbose units occupy sites 2 and 3.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 325, 1061-1076) copyright 2003.

Figures were selected by an automated process.