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large solvent channels and the position of the active site. The x-ray analysis confirmed the trimeric
nature of the enzyme, as the subunits are related by the crystallographic three-fold axis.
Page 159
A ribbon diagram of the trimer is shown in Figure 4B. Each monomer contains an eight-stranded β sheet
and a five-stranded β sheet that join to form a distorted β barrel. Seven α helices surround this β sheet
structure.
The active site is an irregular indentation on the surface of the enzyme, located from the position of a
tightly bound sulfate ion and various substrate analogs. These investigations revealed the identity of the
exact amino acids constituting the active site region; such detail was a prerequisite to drug design.
Information of greater import emerged from analyses of the complexes formed when synthetic
nucleosides, including previously discovered inhibitors, were diffused into the active site.
B. The Active Site
The structural determinations also yielded a surprise. The shape of the enzyme changes when a purine is
bound. The famous lock-and-key analogy [20] has a fallacy; the shape of the lock is not static, but
flexible. Awareness of these conformational changes critically aided our modeling efforts, allowing
prediction of which parts of PNP could change shape to interact with a proposed inhibitor.
A “swinging gate” consisting of residues 241–260 controls access to the active site (Figure 4C). These
residues in the native structure had poorly defined electron density with high thermal motion. The gate
opens in the native enzyme to accommodate the substrate or inhibitor. The maximum movement caused
by substrate or inhibitor binding occurs at His 257, which is displaced outwards by several angstroms.
After binding, the electron density becomes well defined. The gate is anchored near the central β sheet
at one end and near the C-terminal helix at the other end. The gate movement is complex and appears to
involve a helical transformation near residues 257–261.
Consequently, initial inhibitor modeling attempts using the native PNP structure were far less successful
than subsequent analyses in which coordinates for the guanine-PNP complex were used. Because of the
magnitude of the changes that occur during substrate binding, it is unlikely that modeling studies based
on the native structure alone would have accurately predicted the structure of PNP/inhibitor complexes.
The active site is located near the subunit-subunit boundary within the trimer and involves seven
polypeptide segments from one subunit and a short loop from the adjacent subunit (Figure 4D). The
purine binding site employs residues Glu 201, Lys 244, and Asn 243 to form hydrogen bonds with N1,
O6, and N7 of purine. The remainder of the purine binding pocket is largely
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