Crystal structures of soybean β-amylase reacted with β-maltose and maltal: Active site components and their apparent roles in catalysis

Bunzo Mikami, Massimo Degano, Edward J. Hehre, James C. Sacchettini

Research output: Contribution to journalArticlepeer-review

Abstract

The crystal structures of catalytically competent soybean β-amylase, unliganded and bathed with small substrates (β-maltose, maltal), were determined at 1.9-2.2-Å resolution. Two molecules of β-maltose substrate bind to the protein in tandem, with some maltotetraose enzymic condensation product sharing the same binding sites. The β-amylase soaked with maltal shows a similar arrangement of two bound molecules of 2-deoxymaltose, the enzymic hydration product. In each case the nonreducing ends of the saccharide ligands are oriented toward the base of the protein's active site pocket. The catalytic center located between the bound disaccharides and found deeper in the pocket than where the inhibitor α-cyclodextrin binds, is characterized by the presence of oppositely disposed carboxyl groups of two conserved glutamic acid residues. The OE2 carboxyl of Glu 186 is below the plane of the penultimate glucose residue (Glc 2) of bound maltotetraose, 2.6 Å from the oxygen atom of that ligand's penultimate α-1,4-glucosidic linkage The OE2 carboxyl of Glu 380 lies above the plane of Glc 2, 2.8 Å from the O-1 atom of the more deeply bound β-maltose. Saccharide binding does not alter the spatial coordinates of these two carboxyl groups or the overall conformation of the 57-kDa protein. However, the saccharide complexes of the active enzyme are associated with a significant (10 Å) local conformational change in a peptide segment of a loop (L3) that borders the active site pocket. This "hinged" loop is in an open position, extending into solvent in the unliganded protein (and the solvent channel in the crystal) and in a closed position, forming part of the active site (through interactions of Asp 101 with Glc 1 and of Val 99 with Glc 4) in the enzyme/ saccharide complexes. With loop 3 closed, van der Waals interactions between the methyl groups of Val 99 and those of Leu 383 shield the catalytic groups and the reaction center from solvent, presumably allowing an ordered water molecule located near Glu 380 to provide topological control of the steric outcome of hydrolysis/hydration reactions. Loop 3 in the open position is essential for reaction product release and departure.

Original languageEnglish
Pages (from-to)7779-7787
Number of pages9
JournalBiochemistry
Volume33
Issue number25
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry

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