3CPU

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

Summary for 3CPU

• Entry DOI: 10.2210/pdb3cpu/pdb
• Related PRD ID: PRD_900001
• Descriptor: Pancreatic alpha-amylase, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: amylase, acarbose, mutagenesis, diabetes, catalysis, pancreatic, enzyme, human, hydrolase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 1
• Total formula weight: 56348.15

Authors

Brayer, G.D.,Sidhu, G.,Maurus, R.,Rydberg, E.H.,Braun, C.,Wang, Y.,Nguyen, N.T.,Overall, C.M.,Withers, S.G. (deposition date: 1999-06-08, release date: 2001-06-30, Last modification date: 2024-10-30)

Primary citation

Brayer, G.D.,Sidhu, G.,Maurus, R.,Rydberg, E.H.,Braun, C.,Wang, Y.,Nguyen, N.T.,Overall, C.M.,Withers, S.G.
Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.
Biochemistry, 39:4778-4791, 2000

PubMed Abstract: 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.

PubMed: 10769135
DOI: 10.1021/bi9921182

Experimental method

X-RAY DIFFRACTION (2 Å)