Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad

• Published in: Glycobiology (Oxford) 1995-02, Vol.5 (1), p.45-50
• Main Authors: Mirkov, T.E, Evans, S.V, Wahlstrom, J, Gomez, L, Young, N.M, Chrispeels, M.J
• Format: Article
• Language: English
• Subjects: active site; alpha-amylase; alpha-Amylases - antagonists & inhibitors; Amino Acid Sequence; amino acid sequences; Arginine; Base Sequence; Binding Sites; comparisons; complementary DNA; enzyme inhibitors; Fabaceae - metabolism; glycoproteins; Molecular Sequence Data; Mutagenesis, Site-Directed; mutagensis; mutants; Nicotiana; Oligodeoxyribonucleotides; Phaseolus vulgaris; Phytohemagglutinins - chemistry; Pisum sativum - metabolism; Plant Lectins; Plant Proteins - chemistry; Plant Proteins - pharmacology; Plants, Medicinal; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; seeds; Seeds - metabolism; Sequence Homology, Amino Acid; site-directed mutagenesis; tendamistat; transgenic plants; Trypsin Inhibitors; Tryptophan; Tyrosine; α-amylase
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/5.1.45

Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein α-amylase inhibitor (αAI). Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein αAI, which exerts its biological activity by making a 1:1 complex with α-amylase. α-Amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn77 to form an active molecule. Comparision of the amino acid sequence of the bean αAI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp188 might be involved in the inhibitory site. When a three-dimensional model of the bean αAI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn77. To test this site hypothesis, mutants of αAI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco. The mutant proteins R74N and WSY18–190GNV, as well as the double mutant, were inactive as inhibitors. These findings suggest that the active site of αAI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77may be necessary to bring these residues in close proximity.