We report a multifaceted study of the active site region of human pancreatic
-amylase. Througha series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, anoverall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage modeoccurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are threesugars 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 theimportance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kineticand structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution ofthis residue leading to an ~10
6-fold drop in catalytic activity. Structural studies show that the originalpseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series ofhydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spansthe 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 theinhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectivelyin the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitutionof the side chains of either Glu233 or Asp300 leads to as much as a ~10
3-fold decrease in catalyticactivity. Structural analyses of the Asp300Asn variant of human pancreatic
-amylase and its complexwith acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.