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| - | [[Image:1nm9.jpg|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1nm9| PDB=1nm9 | SCENE= }} | | {{STRUCTURE_1nm9| PDB=1nm9 | SCENE= }} |
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| - | '''Crystal structure of recombinant human salivary amylase mutant W58A'''
| + | ===Crystal structure of recombinant human salivary amylase mutant W58A=== |
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| - | ==Overview==
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| - | 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. | + | The line below this paragraph, {{ABSTRACT_PUBMED_15182367}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15182367 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_15182367}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Catalysis]] | | [[Category: Catalysis]] |
| | [[Category: Tim barrel]] | | [[Category: Tim barrel]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 02:42:10 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 29 13:01:36 2008'' |
Revision as of 10:01, 29 July 2008
Template:STRUCTURE 1nm9
Crystal structure of recombinant human salivary amylase mutant W58A
Template:ABSTRACT PUBMED 15182367
About this Structure
1NM9 is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
Reference
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity., Ramasubbu N, Ragunath C, Mishra PJ, Thomas LM, Gyemant G, Kandra L, Eur J Biochem. 2004 Jun;271(12):2517-29. PMID:15182367
Page seeded by OCA on Tue Jul 29 13:01:36 2008
