DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A POTENT XYLOSE ISOMERASE INHIBITOR, D-THREONOHYDROXAMIC ACID, AND HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX
Kn. Allen et al., DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A POTENT XYLOSE ISOMERASE INHIBITOR, D-THREONOHYDROXAMIC ACID, AND HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX, Biochemistry, 34(11), 1995, pp. 3742-3749
The binding of a potent inhibitor to the enzyme D-xylose isomerase fro
m Streptomyces olivochromogenes was examined by kinetics and X-ray cry
stallography. The inhibitor D-threonohydroxamic acid (THA) was designe
d to mimic the putative transition state of the isomerization step cat
alyzed by the enzyme on the substrate xylose. THA was synthesized and
found to be a slow-binding competitive inhibitor with the substrate gl
ucose. The K-i less than or equal to 100 nM was at least one million-f
old less than the K-M for glucose. The X-ray crystallographic structur
e of xylose isomerase with THA soaked into the crystals (concentration
= 1000K(i)) was obtained to 1.6-Angstrom resolution and refined to an
R factor of 21.6%. The free enzyme and the enzyme in the xylose isome
rase-THA complex show no significant structural differences. THA binds
in an analogous fashion to glucose, in a linear conformation, forming
ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182.
On the basis of these similarities to glucose binding and its potent i
nhibition, we propose that THA resembles the transition state for the
enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms
a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do
so. By analogy, we propose that, during the catalytic reaction, C2 of
the substrate glucose is deprotonated, and that this proton can be mo
ved to the C1 hydroxyl concomitant with hydride transfer. We find evid
ence for metal movement during catalysis upon deprotonation of the C2
hydroxyl, to allow formation of a bridging ligand. Ln addition, the ob
servation in the xylose isomerase-THA complex of a water molecule boun
d to Mg-2 instead of a hydroxide (as seen in the native structure) sug
gests that protonation of the hydroxyl occurs in a step following ring
opening. The metal-bound hydroxide ion may act as a general base to d
eprotonate the C2 hydroxyl of the substrate. Due to their ability to a
ct as metal ligands, hydroxamic acids may be general inhibitors of dim
etallic enzymes.