Role of the Divalent Metal Ion in Sugar Binding, Ring Opening, and Isomerization by D-Xylose Isomerase: Replacement of a Catalytic Metal by an Amino Acid

Abstract

The distinct roles of the two magnesium ions essential to the activity of D-xylose isomerase from Streptomyces olivochromogenes were examined. The enzyme-magnesium complex was isolated, and the stoichiometry of cation binding determined by neutron activation analysis to be 2 mol of magnesium per mole of enzyme. A plot of Mg2+ added versus Mg2+ bound to enzyme is consistent with apparent KD values of < or = 0.5-1.0 mM for one Mg2+ and < or = 2-5 mM for the second. A site-directed mutant of D-xylose isomerase was designed to remove the tighter, tetracoordinated magnesium binding site (site 1, Mg-1); Glu180 was replaced with Lys180. The stoichiometry of metal binding to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ring-opening assays with 1-thioglucose (H2S released upon ring opening) show E180K catalyzes the opening of the sugar ring at 20% the rate of the wild-type, but E180K does not catalyze isomerization of glucose to fructose. Thus, the magnesium bound to Glu180 is essential for isomerization but not essential for ring opening. The X-ray crystallographic structures of E180K in the absence of magnesium and in the presence and absence of 250 mM glucose were obtained to 1.8-A resolution and refined to R factors of 17.7% and 19.7%, respectively. The wild-type and both E180K structures show no significant structural differences, except the epsilon-amino group of Lys180, which occupies the position usually occupied by the Mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms and Free Energies of Enzymatic Reactions

“…A number of XyI structures with the substrate D-glucose or transition state analogues have been determined. 147,[155][156][157][158][159] Despite very strong binding by the inhibitor, which can potentially induce large protein conformational changes, Petsko and co-workers showed that the overall structural difference between the inhibitor complex and the apoenzyme had a very small C α RMS deviation of 0.27 Å, suggesting such a global comparison of structures was insufficient for characterizing active-site interactions. 147,156 Two structures are compared in the present discussion, the D-glucose complex (1XYB) determined by Whitlow et al 159 and the inhibitor complex (2GYI) determined by Allen et al, 147,156 which leads to a small difference in the proposed coordination sphere to the Mg2 ion (i.e., the second Mg ion cofactor), which is a "mobile" ion during the reaction (Scheme 3).…”

“…159 An important feature of the active site of XyI is a combination of two divalent (Mg 2+ , Co 2+ , Mn 2+ ) ions bridged by an aspartate residue; 250 a growing number of enzymes are now known to share this structural motif. 155,156 The overall enzymatic process is rather complex, involving the opening of the pyranose sugar ring, isomerization by a hydride transfer mechanism, and reclosing to form the cyclic sugar product. 159 X-ray structures have captured a number of intermediate configurations using various substrate and transition state analogue inhibitors.…”

Protein-Carbohydrate Interactions

“…203 Allen, K. N., Lavie, A., Glasfeld, A., Tanada, T. N., Gerrity, D. P., Carlson, S. C., Farber, G. K., Petsko, G. A. and Ringe, D. (1994) Baneijee, R., Das, K., Ravishankar, R., Suguna, K., Surolia, A. and Vijayan, M. (1996) Conformation , Proteins: Struct., Funct., Genet., 12,[203][204][205][206][207][208][209][210][211][212][213][214][215][216][217][218][219][220][221][222] Goldstein, I. J., Reichert, C. M. and Misaki, A. (1974) Stidy of Concanavalin A: The Proton of Asp28, J. Chem.…”

“…The only other direct sugar -metal interaction known is xylose isomerase in which the sugar ligates two Mg^^ ions Lavie et al, 1994). (Naismith et al, 1994;Allen et al, 1994). (Bourne et a l, 1994), pea lectin (Rini et a l, 1993), WGA (Wright, 1992), MBP (Weis et a l, 1992) and the bluebell lectin (SCA) (http://plab.ku.dk/tcbh/Lectinsl2AVright/paper.htm).…”

Restoration of a Defective Lactococcus lactis Xylose Isomerase

“…Although they share only 20 to 30% amino acid sequence identity with group I XIs, the active-site residues in group I and group II enzymes are highly conserved ( Table 1). The structures of monomeric, homodimeric, and homotetrameric XIs from a number of microbial sources including Streptomyces (5-7), Actinoplanes (1,21,29,36), Arthrobacter (37), Bacillus (28), and Thermus (9) were solved by X-ray crystallography. A structural feature of monomeric XIs is that they consist of an (␣/␤) 8 barrel having an active site and a C-terminal loop region.…”