JBC, Vol. 250, Issue 23, 9021-9027, Dec, 1975
Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions
J. M. Young, K. J. Schray and A. S. Mildvan
The interaction of D-xylose isomerase purified from two sources with Mn2+
and D-xylose or the competitive inhibitor xylitol has been examined by
nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound
Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed,
providing independent evidence for the specificity of D-xylose isomerase
for the alpha anomeric form of D-xylose. The exchange rate of
alpha-D-xylose into the ternary complex, determined from the normalized
paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the
carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction
by 3 orders of magnitude. The amount of xylitol necessary to displace
alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with
the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol
previously determined by the methods of enzyme kinetics. These results
suggest that the NMR experiments observe complexes of D-xylose isomerase
which are kinetically and thermodynamically competent to participate in
catalysis. From the frequency dependence of the paramagnetic contribution
to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of
alpha-D-xylose, the correlation time (tauc) which modulates the dipolar
interaction between enzyme-bound Mn2+ and alpha-D-xylose has been
determined (5.1 x 1o(-10) s). From these observations a range of calculated
distances between enzyme-bound Mn2+ and the carbon 1 proton of
alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has
comparable effects on the carbon 1, carbon 2, and carbon 5 protons of
alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate
are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A)
between the enzyme-bound Mn2+ and the terminal methylene protons of
xylitol, an analog of the open chain intermediate in the reaction, has been
determined. The results of the present substrate relaxation and previous
water relaxation studies suggest that two small ligands such as water
molecules or a large portion of the protein intervene between the bound
metal ion and the bound substrate in the active ternary complex.
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