title: Isotopic exchange plus substrate and inhibition kinetics of D-xylose isomerase do not support a proton-transfer mechanism.
abstract: The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-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 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH 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 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-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 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 energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR 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.
authors.affiliation: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
authors.forename: K N
authors.initials: KN
authors.lastname: Allen
authors.forename: A
authors.initials: A
authors.lastname: Lavie
authors.forename: G K
authors.initials: GK
authors.lastname: Farber
authors.forename: A
authors.initials: A
authors.lastname: Glasfeld
authors.forename: G A
authors.initials: GA
authors.lastname: Petsko
authors.forename: D
authors.initials: D
authors.lastname: Ringe
mesh_terms: D019747:Aldose-Ketose Isomerases; D002125:Calcium Gluconate / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005459:Fluorides; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000494:pharmacology; D011522:Protons; D017421:Sequence Analysis; D012997:Solvents; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014316:Tritium
chemical_list: D011522:Protons; D012997:Solvents; D014316:Tritium; D008345:Manganese; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D005947:Glucose; D005459:Fluorides; D002125:Calcium Gluconate