Kn. Allen et al., ISOTOPIC EXCHANGE PLUS SUBSTRATE AND INHIBITION-KINETICS OF D-XYLOSE ISOMERASE DO NOT SUPPORT A PROTON-TRANSFER MECHANISM, Biochemistry, 33(6), 1994, pp. 1481-1487
The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or
Mn2+-dependent enzyme that catalyzes the aldose-ketose isomerization o
f xylose to xylulose or of glucose to fructose. Proton exchange into w
ater during enzyme-catalyzed isomerization of C-2 tritiated glucose at
15, 25 and 55 degrees C shows <0.6% exchange (the loss of one proton
in every billion turnovers). High concentrations of guanidine hydrochl
oride and extremes of pH had no effect on the amount of exchange detec
ted. Such a low percentage of exchange is inconsistent with a proton-t
ransfer mechanism as the main kinetic pathway for isomerization. F-19
NMR experiments showed no release of fluoride after incubation of the
enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fl
uoroallose (both are competitive inhibitors with K-i values of 600 mM)
. This result is also inconsistent with a proton-transfer mechanism. A
hydride-shift mechanism following ring opening has been proposed for
the isomerization. Enzyme-catalyzed ring opening was directly measured
by demonstrating H2S release upon reaction of xylose isomerase with 1
-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose
to fructose exhibited linear Arrhenius behavior with an activation ene
rgy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determini
ng step occurs over this temperature range. C-13 NMR experiments with
glucose show that enzyme-bound magnesium or manganese does not interac
t specifically with any one site on the sugar. These results are consi
stent with nonproductive binding modes for the substrate glucose in ad
dition to productive binding.