3cpu

SUBSITE MAPPING OF THE ACTIVE SITE OF HUMAN PANCREATIC ALPHA-AMYLASE USING SUBSTRATES, THE PHARMACOLOGICAL INHIBITOR ACARBOSE, AND AN ACTIVE SITE VARIANT

spinqualitylabelsall models 3cpu, resolution 2.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3cpu is a 1 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2Å Ligands: CA, CL, GLC, PCA, PRD_900001 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function AMYP_HUMAN 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 We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding. Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.,Brayer GD, Sidhu G, Maurus R, Rydberg EH, Braun C, Wang Y, Nguyen NT, Overall CM, Withers SG Biochemistry. 2000 Apr 25;39(16):4778-91. PMID:10769135 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Amylase 3D structures

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