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| | {{STRUCTURE_2cpu| PDB=2cpu | SCENE= }} | | {{STRUCTURE_2cpu| PDB=2cpu | SCENE= }} |
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| - | '''SUBSITE MAPPING OF THE ACTIVE SITE OF HUMAN PANCREATIC ALPHA-AMYLASE USING SUBSTRATES, THE PHARMACOLOGICAL INHIBITOR ACARBOSE, AND AN ACTIVE SITE VARIANT'''
| + | ===SUBSITE MAPPING OF THE ACTIVE SITE OF HUMAN PANCREATIC ALPHA-AMYLASE USING SUBSTRATES, THE PHARMACOLOGICAL INHIBITOR ACARBOSE, AND AN ACTIVE SITE VARIANT=== |
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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_10769135}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 10769135 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_10769135}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Mutagenesis]] | | [[Category: Mutagenesis]] |
| | [[Category: Pancreatic]] | | [[Category: Pancreatic]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 22:45:45 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 08:22:09 2008'' |
Revision as of 05:22, 28 July 2008
Template:STRUCTURE 2cpu
SUBSITE MAPPING OF THE ACTIVE SITE OF HUMAN PANCREATIC ALPHA-AMYLASE USING SUBSTRATES, THE PHARMACOLOGICAL INHIBITOR ACARBOSE, AND AN ACTIVE SITE VARIANT
Template:ABSTRACT PUBMED 10769135
About this Structure
2CPU is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
Reference
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
Page seeded by OCA on Mon Jul 28 08:22:09 2008
