Linked Life Data

12527308

Source:http://linkedlifedata.com/resource/pubmed/id/12527308

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2003-1-15
pubmed:databankReference
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0022-2836
pubmed:author
pubmed:issnType
Print
pubmed:day
31
pubmed:volume
325
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1061-76
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:12527308-Acarbose, pubmed-meshheading:12527308-Amylases, pubmed-meshheading:12527308-Binding Sites, pubmed-meshheading:12527308-Carbohydrate Conformation, pubmed-meshheading:12527308-Catalysis, pubmed-meshheading:12527308-Crystallography, X-Ray, pubmed-meshheading:12527308-Humans, pubmed-meshheading:12527308-Hydrolysis, pubmed-meshheading:12527308-Kinetics, pubmed-meshheading:12527308-Ligands, pubmed-meshheading:12527308-Maltose, pubmed-meshheading:12527308-Models, Molecular, pubmed-meshheading:12527308-Molecular Structure, pubmed-meshheading:12527308-Mutagenesis, Site-Directed, pubmed-meshheading:12527308-Oligosaccharides, pubmed-meshheading:12527308-Protein Conformation, pubmed-meshheading:12527308-Salivary Glands, pubmed-meshheading:12527308-Starch, pubmed-meshheading:12527308-Substrate Specificity
pubmed:year
2003
pubmed:articleTitle
Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase.
pubmed:affiliation
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA. n.ramasubbu@umdnj.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.