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

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 less than or equal to 0.5-1.0 mM for one Mg2+ and less than or equal to 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-Angstrom 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-l. Other active-site residues usually bound to Mg-1 are only slightly changed in position, with a magnesium ion occupying the second metal-binding site (site 2, Mg-2). Structurally, the lysine residue has successfully replaced Mg-1. Europium(III) excitation spectroscopy shows that when Eu3+ is bound to E180K at site 2 (in the absence of substrate), one to two water molecules is coordinated to Eu3+. The X-ray crystallographic structure of the E180K enzyme shows that one hydroxide molecule is coordinated to Mg-2. This result is consistent with the apparent pK(a) of 7.2 from a plot of pH versus log V-max/K-m for the wild-type enzyme. Hydroxide coordinated to Mg-2 could act to deprotonate the glucose O2 and protonate O1 in a step concomitant with the hydride-transfer step of isomerization.