@article { ISI:000086737700025,
	title = {Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques},
	journal = {BIOCHEMISTRY},
	volume = {39},
	number = {16},
	year = {2000},
	month = {APR 25},
	pages = {4778-4791},
	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-origosaccharyl 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 gIycon 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{{\textquoteright}{\textquoteright}} 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 similar to 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 {\textquoteleft}{\textquoteleft}glycosidic{{\textquoteright}{\textquoteright}} 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 similar to 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.},
	issn = {0006-2960},
	doi = {10.1021/bi9921182},
	author = {BRAYER, GD and Sidhu, G and Maurus, R and Rydberg, EH and BRAUN, C and WANG, YL and Nguyen, NT and Overall, CH and Withers, SG}
}
