GrainGenes Reference Report: JBC-268-22480
Reference
JBC-268-22480
Title
Site-directed mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley alpha-amylase 1
Journal
Journal of Biological Chemistry
Year
1993
Volume
268
Pages
22480-22484
Author
Sogaard M Kadziola A Haser R Svensson B
Abstract
The Pseudotetrasaccharide acarbose has high affinity for the active site (Ki,app = 1 micromolar) and low affinity for a secondary site (Kd = 2.3 mm) in barley alpha-amylase 1, distinguished by inhibition kinetics and spectral perturbation Mutants of putative catalytic residues, D180N, E205Q, and D291N, are inactive and display low affinity for acarbose-Sepharose H93N and H29ON mutants, at invariant residues, have kcat/Km for p-nitrophenylmaltoheptaoside of 0.3 and 1.2% of wildtype A corresponding 370- and 85-fold increased Ki,app for acarbose and a lack of shifts in pH activity profiles indicate that these histidines participate in transition state stabilization but not directly in catalysis This finding agrees with H bonding to OH groups of the valienamine ring of acarbose in the three-dimensional structure. Loss of inhibition above pH 6 supports that acarbose is most potent in protonated form. The low affinity site contains Trp(278) and Trp(279), known to bind cyclomaltoheptaose. While the W279A mutant has 10- fold decreased affinity for starch granules, production of Trp(278) mutants failed. The invariant Trp(278) is perhaps critical for stability or folding in cereal alpha-amylases.
Keyword
GrainGenes is a product of the Agricultural Research Service of the US Department of Agriculture.
GrainGenes Reference Report: JBC-268-22480
|
| |||||
|
| |||||
|
| |||||
|
| |||||
|
| |||||
|
| |||||
|
| |||||
|
| |||||
|
GrainGenes is a product of the Agricultural Research Service of the US Department of Agriculture. | |||