|
|
| Line 1: |
Line 1: |
| [[Image:3cpu.jpg|left|200px]] | | {{Seed}} |
| | [[Image:3cpu.png|left|200px]] |
|
| |
|
| <!-- | | <!-- |
| Line 9: |
Line 10: |
| {{STRUCTURE_3cpu| PDB=3cpu | SCENE= }} | | {{STRUCTURE_3cpu| PDB=3cpu | SCENE= }} |
|
| |
|
| '''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=== |
|
| |
|
|
| |
|
| ==Overview==
| | <!-- |
| 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. |
| | --> |
| | {{ABSTRACT_PUBMED_10769135}} |
|
| |
|
| ==About this Structure== | | ==About this Structure== |
| Line 40: |
Line 44: |
| [[Category: Mutagenesis]] | | [[Category: Mutagenesis]] |
| [[Category: Pancreatic]] | | [[Category: Pancreatic]] |
| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 21:57:14 2008'' | | |
| | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Jul 27 23:29:46 2008'' |
