Subsite Mapping of the Human Pancreatic -Amylase Active Site through Structural, Kinetic, and Mutagenesis Techniques
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- 作者：Gary D. Brayer ; Gary Sidhu ; Robert Maurus ; Edwin H. Rydberg ; Curtis Braun ; Yili Wang ; Nham T. Nguyen ; Christopher M. Overall ; and Stephen G. Withers
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：April 25, 2000
- 年：2000
- 卷：39
- 期：16
- 页码：4778 - 4791
- 全文大小：329K
- 年卷期：v.39,no.16(April 25, 2000)
- ISSN：1520-4995
文摘
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 ~106-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 ~103-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.
-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 ~106-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 ~103-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.