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New crystallographic findings are presented which offer a deeper understanding
of the structure and functioning of beta-amylase, the first known exo-type
starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean
beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been
determined at 2.0-A resolution with a conventional R-value of 17.5%. The model
contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1
alpha-cyclodextrin molecule. The protein consists of a core with an
(alpha/beta)8 supersecondary structure, plus a smaller globular region formed by
long loops (L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta
5. Between the two regions is a cleft that opens into a pocket whose floor
contains the postulated catalytic center near the carboxyl group of Glu 186. The
annular alpha-cyclodextrin binds in (and partly projects from) the cleft with
its glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1
--> 4) glucosidic linkage progression running clockwise as viewed from that
side. The ligand does not bind deeply enough to interact with the carboxyl group
of Glu 186. Rather, it occupies most of the cleft entrance, strongly suggesting
that alpha-cyclodextrin inhibits catalysis by blocking substrate access to the
more deeply located reaction center. Of the various alpha-cyclodextrin
interactions with protein residues in loops L4, L5, L6, and L7, most notable is
the shallow inclusion complex formed with Leu 383 (in L7, on the core side of
the cleft) through contacts of its methyl groups with the C-3 atoms of four of
the ligand's D-glucopyranosyl residues. All six residues of the bound
alpha-cyclodextrin are of 4C1 conformation and are joined by alpha-1,4 linkages
with similar torsional angles to form a nearly symmetrical torus as reported for
crystalline inclusion complexes with alpha-cyclodextrin. We envision a
significant role for the methyl groups of Leu 383 at the cleft entrance with
respect to the productive binding of the outer chains of starch.
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