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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 D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O 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 (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the 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) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Toteva MM,Silvaggi NR,Allen KN,Richard JP

doi

10.1021/bi201378c

subject

Has Abstract

pub_date

2011-11-22 00:00:00

pages

10170-81

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde.