The pseudotetrasaccharide acarbose has high affinity for the active site (Ki,app = 1 microM) 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 H290N mutants, at invariant residues, have kcat/Km for p-nitrophenylmaltoheptaoside of 0.3 and 1.2% of wild-type. 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 Trp278 and Trp279, known to bind cyclomaltoheptaose. While the W279A mutant has 10-fold decreased affinity for starch granules, production of Trp278 mutants failed. The invariant Trp278 is perhaps critical for stability or folding in cereal alpha-amylases.
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1993|