LOCATION OF THE ACTIVE-SITE OF THE BEAN ALPHA-AMYLASE INHIBITOR AND INVOLVEMENT OF A TRP, ARG, TYR TRIAD

Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-amylase inhibitor (alpha AI), Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein alpha AI, which exerts its biological activity by making a 1:1 complex with alpha-amylase. alpha-Amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn(77) to form an active molecule, Comparison of the amino acid sequence of the bean alpha AI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp(188) might be involved in the inhibitory site, When a three-dimensional model of the bean alpha AI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn(77). To test this site hypothesis, mutants of alpha AI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco, The mutant proteins R74N and WSY188-190GNV, as well as the double mutant, were inactive as inhibitors, These findings suggest that the active site of alpha AI consists of W-188, R(74) and Y-190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn(77) may be necessary to bring these residues in close proximity.