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: phytohaema gglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-a mylase inhibitor (alpha AI), Whereas the active site of lectins has be en 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 i s synthesized as a 30 kDa precursor glycoprotein that needs to be proc essed at Asn(77) to form an active molecule, Comparison of the amino a cid sequence of the bean alpha AI with that of the bacterial amylase i nhibitor, tendamistat, suggested that a region around Trp(188) might b e involved in the inhibitory site, When a three-dimensional model of t he bean alpha AI was constructed based on its homology to the legume l ectins, this Trp region was alongside Asn(77). To test this site hypot hesis, mutants of alpha AI were created by site-directed mutagenesis o f the cDNA and expressed in transgenic tobacco, The mutant proteins R7 4N and WSY188-190GNV, as well as the double mutant, were inactive as i nhibitors, These findings suggest that the active site of alpha AI con sists of W-188, R(74) and Y-190, in analogy to the Trp-Arg-Tyr motif o f tendamistat, and that the processing of the polypeptide at Asn(77) m ay be necessary to bring these residues in close proximity.