3ij9

Directed 'in situ' Elongation as a Strategy to Characterize the Covalent Glycosyl-Enzyme Catalytic Intermediate of Human Pancreatic a-Amylase

spinqualitylabelsall models 3ij9, resolution 1.85Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3ij9 is a 1 chain structure with sequence from Human. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: B0D, B9D, BGC, CA, CL NonStd Res: PCA Related: 3ij7, 3ij8 Gene: AMY2A (HUMAN) Activity: Alpha-amylase, with EC number 3.2.1.1 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed While covalent catalytic intermediates of retaining alpha-transglycosylases have been structurally characterized previously, no such information for a hydrolytic alpha-amylase has been obtained. This study presents a new "in situ" enzymatic elongation methodology that, for the first time, has allowed the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic alpha-amylase. This has been achieved by the use of a 5-fluoro-beta-l-idosyl fluoride "warhead" in conjunction with either alpha-maltotriosyl fluoride or 4'-O-methyl-alpha-maltosyl fluoride as elongation agents. This generates an oligosaccharyl-5-fluoroglycosyl fluoride that then reacts with the free enzyme. The resultant covalent intermediates are extremely stable, with hydrolytic half-lives on the order of 240 h for the trisaccharide complex. In the presence of maltose, however, they undergo turnover via transglycosylation according to a half-life of less than 1 h. Structural studies of intermediate complexes unambiguously show the covalent attachment of a 5-fluoro-alpha-l-idosyl moiety in the chair conformation to the side chain of the catalytic nucleophile D197. The elongated portions of the intermediate complexes are found to bind in the high-affinity -2 and -3 binding subsites, forming extensive hydrogen-bonding interactions. Comparative structural analyses with the related noncovalent complex formed by acarbose highlight the structural rigidity of the enzyme surface during catalysis and the key role that substrate conformational flexibility must play in this process. Taken together, the structural data provide atomic details of several key catalytic steps. The scope of this elongation approach to probe the active sites and catalytic mechanisms of alpha-amylases is further demonstrated through preliminary experiments with porcine pancreatic alpha-amylase. Directed "in Situ" Inhibitor Elongation as a Strategy To Structurally Characterize the Covalent Glycosyl-Enzyme Intermediate of Human Pancreatic alpha-Amylase (,).,Zhang R, Li C, Williams LK, Rempel BP, Brayer GD, Withers SG Biochemistry. 2009 Oct 15. PMID:19803533 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Amylase 3D structures

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