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

The pseudotetrasaccharide acarbose has high affinity for the active si te (K(i,app) = 1 muM) and low affinity for a secondary site (K(d) = 2. 3 mM) in barley alpha-amylase 1, distinguished by inhibition kinetics and spectral perturbation. Mutants of putative catalytic residues, D18 0N, E205Q, and D291N, are inactive and display low affinity for acarbo se-Sepharose. H93N and H290N mutants, at invariant residues, have k(ca t)/K(m) for p-nitrophenylmaltoheptaoside of 0.3 and 1.2% of wild-type. A corresponding 370- and 85-fold increased K(i,app) for acarbose and a lack of shifts in pH activity profiles indicate that these histidine s participate in transition state stabilization but not directly in ca talysis. This finding agrees with H bonding to OH groups of the valien amine ring of acarbose in the three-dimensional structure. Loss of inh ibition 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 affi nity for starch granules, production of Trp278 mutants failed. The inv ariant Trp278 is perhaps critical for stability or folding in cereal a lpha-amylases.