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 pancreati c alpha-amylase. Through a series of novel kinetic analyses using malto-oli gosaccharides and malto-origosaccharyl fluorides, an overall cleavage actio n pattern for this enzyme has been developed. The preferred binding/cleavag e 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) site s. Overall it appears that five binding subsites span the active site, alth ough an additional gIycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of subst rates modified at the 2 and 4" positions also highlight the importance of t hese 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 s imilar to 10(6)-fold drop in catalytic activity. Structural studies show th at the original pseudo-tetrasaccharide structure of acarbose is modified up on binding, presumably through a series of hydrolysis and transglycosylatio n reactions. The end result is a pseudo-pentasaccharide moiety that spans t he active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp3 00, along with a water molecule, are aligned about the inhibitor N-linked g lycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. I ndeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a similar to 10(3)-fold decrease in c atalytic activity. Structural analyses of the Asp300Asn variant of human pa ncreatic alpha-amylase and its complex with acarbose clearly demonstrate th e importance of Asp300 to the mode of inhibitor binding.