PDBsum entry: 1jxj

Hydrolase

PDB id: 1jxj

Contents

Protein chain

496 a.a. *

Metals

_CL

_CA

Waters ×294

* Residue conservation analysis

PDB id:

1jxj

Name:

Hydrolase

Title:

Role of mobile loop in the mechanism of human salivary amylase

Structure:

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

Source:

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

Resolution:

1.99Å R-factor: 0.166 R-free: 0.198

Authors:

N.Ramasubbu,C.Ragunath,Z.Wang,P.J.Mishra,L.M.Thomas

Key ref:

N.Ramasubbu et al. (2004). Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity. Eur J Biochem, 271, 2517-2529. PubMed id: 15182367 DOI: 10.1111/j.1432-1033.2004.04182.x

Date:

07-Sep-01 Release date: 14-Sep-01

Protein chain

P04745  (AMY1_HUMAN) -  Alpha-amylase 1

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 (1 term)
• Biological process: metabolic process (3 terms)
• Biochemical function: catalytic activity (7 terms)

DOI no: 10.1111/j.1432-1033.2004.04182.x

Eur J Biochem 271:2517-2529 (2004)

PubMed id: 15182367

Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, L.Kandra.

ABSTRACT

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.

Selected figure(s)

Figure 5.
Fig. 5. Difference electron density maps(omit maps) in the mutants W58L and W58A/acarbose complex.(A) Stereodrawing of the 2Fo-Fc omit maps corresponding to residues 58 and 59 in the mutant W58L. (B) Stereodrawing of the 2Fo-Fc omitting density maps corresponding to the bound oligosaccharide in W58A. This complex is made up of a trisaccharide and is named according to subsite location. The electron density map has been contoured at 1 and the final refined coordinates of the corresponding oligosaccharide residues are overlaid.

Figure 7.
Fig. 7. Comparison of the conformation of the bound pseudooligosaccharide in human and fungal -amylases. Superposition of the glycone subsites in HSAmy (thick lines; PDB Code 1mfv) and TAKA-amylase (thin lines; PDB Code 7taa) along with the bound pseudooligosaccharide. These two structures were superposed by fitting the spatial location of the three catalytic residues (equivalent to Asp197, Glu233 and Asp300 in HSAmy). The two bound pseudooligosaccharides (HSAmy vs. TAKA-amylase) traverse two different paths beyond the subsite –2 as shown by the dotted spheres. Note that Trp58 and Trp59 of HSAmy would encounter severe steric interaction if the bound oligosaccharide in HSAmy adopted a conformation as in TAKA-amylase.

The above figures are reprinted by permission from the Federation of European Biochemical Societies: Eur J Biochem (2004, 271, 2517-2529) copyright 2004.

Figures were selected by an automated process.