PMID: 10769135

 

    Legend: Gene, Sites

Title : Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques

Abstract :

1. We report a multifaceted study of the active site region of human pancreatic alpha-amylase
2. 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
3. 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
4. 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
5. 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
6. 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
7. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions
8. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond position ed at the normal site of cleavage
9. 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
10. 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
11. 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

Output (sent_index, trigger, protein, sugar, site):

• 3. sugars, , -, the glycon (-1, -2, -3) sites, sites

Output(Part-Of) (sent_index, protein, site):

• 0. pancreatic alpha-amylase, site
• 1. pancreatic alpha-amylase, region

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX
pancreatic alpha-amylase	279	0,1,11