Binding Energy and Catalysis by d-Xylose Isomerase: Kinetic, Product, and X-ray Crystallographic Analysis of Enzyme-Catalyzed Isomerization of (R)-Glyceraldehyde

Abstract

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and D-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D2O was monitored by 1H NMR spectroscopy and kcat/Km = 0.034 M−1 s−1 was determined for this isomerization at pD 7.0. This is similar to kcat/Km = 0.017 M−1 s−1 for the TIM-catalyzed carbon deprotonation reaction of DGA in D2O at pD 7.0 [Amyes, T. L.; O’Donoghue, A. C. and Richard J. P. (2001) J. Am. Chem. Soc. 123, 11325–11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (kcat/Km = 490 M−1 s−1) than for the TIM-catalyzed isomerization of DGAP (kcat/Km = 9.6 x 106 M−1 s−1) is due to: (i) The larger barrier to conversion of cyclic D-xylose to the reactive linear sugar (5.4 kcal/mol) than for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol). (ii) The smaller intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219–410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) than of the phosphodianion group of DGAP (ca. 12 kcal/mol). The XI-catalyzed isomerization of DGA in D2O at pD 7.0 gives a 90% yield of [1-1H]-DHA and a 10% yield of [1-2H]-DHA, the product of isomerization with deuterium incorporation from solvent D2O. By comparison, the transfer of 3H from labeled hexose substrate to solvent is observed only once in every 109 turnovers for the XI-catalyzed isomerization of [2-3H]-glucose in H2O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994), Biochemistry 33, 1481–1487]. We propose that truncation of the terminal ethylene glycol fragment of D-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high resolution (0.97 Å) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI•sugar complexes.

Insights into Thiol–Aromatic Interactions: A Stereoelectronic Basis for S–H/π Interactions

“…In the structure of the titin domain M7 (Figure 13a), 32 Cys79 engages in an S–H/π interaction with Trp40, with the thiol hydrogen clearly observable in the electron density map. In the structure of D-xylose isomerase (Figure 13b), 33 Cys306 is located within a cage of three aromatic residues (Phe13, Phe286, Phe288), with no traditional hydrogen bond acceptors within interaction range. In the structure of the N-terminal domain of LIP5 (Figure 13c), 34 Cys87 engages in a tight S–H/π interaction with Phe131 (2.63 Å H...C aromatic distance, based on the 1.20 Å S–H bond length and 109.0˚ C–S–H bond angle in the PDB coordinates; the H...C distance would be shorter if modeled with the more typical 1.34 Å S–H bond length and 95˚–100˚ C–S–H bond angle).…”

“…The electron density map is unclear about the hydrogen position, though no traditional hydrogen bond acceptor is near the thiol. 33 (c) Cys87-Phe181 S–H/π interaction (2.63 Å H...C aromatic distance) in pdb 4txr (1.00 Å resolution). 34 Notably, all S–H bonds in the pdb files of (a)–(c) included non-canonical 1.20 Å S–H bond lengths and 109.0˚ C–S–H bond angles.…”

A Paradigm for Enzyme-Catalyzed Proton Transfer at Carbon: Triosephosphate Isomerase

“…An engineered monomeric variant of Tbb TIM (monoTIM) (46) catalyzes isomerization of GAP with k cat / K m = 1000 M −1 s −1 (43) that is similar to k cat / K m for isomerization of xylose catalyzed by xylose isomerase (XI) (47), but much smaller than k cat / K m = 10 7 M −1 s −1 for wildtype Tbb TIM (43). MonoTIM shows no detectable phosphite activation (43).…”

Hydroxide Once or Twice? A combined Neutron Crystallographic and Quantum Chemical Study of the Hydride Shift in D-Xylose Isomerase

“…This minimalist approach is possible due to the 3KCL and 3KCO configurational basis provided by the neutron/X-ray crystallography of Kovalevsky, Langan, Glusker and co-workers [19]. From this structural work, we know that the shoe-box is rigid under the perturbation of ring opening; we also have ion-pair charge states and also orientation of the internal water network, although experimental characterization of the catalytic water remains enigmatic; based on Models I and III, and hints from the difference FO-FC electron density maps in several ultra-high resolution X-ray structures [3,4,31], we conjecture that multiple states (A/B or a/b/c) exist for H 2 O cat .…”

“…Xylose Isomerase (XI) is responsible for aldose to ketose isomerization of hexose and pentose sugars via a hydride shift mechanism [1,2], and is one of the slowest enzymes known, remaining 5 orders of magnitude slower than triosephosphate isomerase that employs the ene-diol mechanism to catalyze a similar aldose to ketose conversion of triose sugars [3,4]. Despite its structural elucidation 25 years ago [5], its current industrial relevance [6,7], and the potential impact on the production of lignocellulosic ethanol [8][9][10][11], efforts to improve the activity of XI have yielded results primarily through enhancement of binding and stability [12][13][14][15], rather than through acceleration of the catalytic rate k cat .…”