ISOTOPIC EXCHANGE PLUS SUBSTRATE AND INHIBITION-KINETICS OF D-XYLOSE ISOMERASE DO NOT SUPPORT A PROTON-TRANSFER MECHANISM

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.