| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-amylase
1 Department of Biochemistry and Molecular Biology,
2 Department of Chemistry, and
3 Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada
4 Novozymes, DK-2880 Bagsvaerd, Denmark
(RECEIVED August 28, 2004; FINAL REVISION November 3, 2004; ACCEPTED November 4, 2004)
The mechanism of allosteric activation of 
-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic -amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible substrate binding loop unique to chloride-dependent -amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent -amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent -amylases.
Keywords: amylase; chloride activation; catalytic activity; acarbose; inhibition; active site; kinetics; X-ray crystallography; protein folding
Abbreviations: HPA, human pancreatic -amylase • CNP-G3, 2-chloro-4-nitrophenyl--maltotrioside • TAKA-amylase, Aspergillus orzyae -amylase • Ani3038, variant Taka-amylase protein containing the mutations W61Q, L250T, and I326R (TAKA-amylase numbering) that was created for this study. Amino acid numbering is according to the sequence alignment presented in Brayer et al. 1995 unless otherwise noted.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041079305.
Reprint requests to: Gary D. Brayer, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; e-mail: brayer{at}interchange.ubc.ca; fax: (604) 822-5227.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Quezada-Calvillo, C. C. Robayo-Torres, A. R. Opekun, P. Sen, Z. Ao, B. R. Hamaker, A. Quaroni, G. D. Brayer, S. Wattler, M. C. Nehls, et al. Contribution of Mucosal Maltase-Glucoamylase Activities to Mouse Small Intestinal Starch {alpha}-Glucogenesis J. Nutr., July 1, 2007; 137(7): 1725 - 1733. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
