Isotopic Exchange plus Substrate and Inhibition Kinetics of D-Xylose Isomerase Do Not Support a Proton-Transfer Mechanism (original) (raw)

The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+-or Mn2+-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15,25 and 55 " C shows <0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of p H had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F N M R experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 m M 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki 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 HIS release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerasecatalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 OC. No change in rate-determining step occurs over this temperature range. 13C N M R experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.