Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques.
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.
Brayer
G D
GD
Department of Biochemistry, University of British Columbia, Vancouver V6T 1Z3, Canada. brayer@laue.biochem.ubc.ca
Sidhu
G
G
Maurus
R
R
Rydberg
E H
EH
Braun
C
C
Wang
Y
Y
Nguyen
N T
NT
Overall
C M
CM
Withers
S G
SG
D020909:Acarbose / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D005459:Fluorides / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013552:Swine; D013816:Thermodynamics; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D008320:Maltose; D000516:alpha-Amylases; D005459:Fluorides; D020909:Acarbose