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
Kn. Allen et al., 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, Biochemistry, 33(6), 1994, pp. 1488-1494
The distinct roles of the two magnesium ions essential to the activity
of D-xylose isomerase from Streptomyces olivochromogenes were examine
d. 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+ boun
d to enzyme is consistent with apparent KD values of less than or equa
l 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 r
emove the tighter, tetracoordinated magnesium binding site (site 1, Mg
-1); Glu180 was replaced with Lys180. The stoichiometry of metal bindi
ng to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ri
ng-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 isomer
ization but not essential for ring opening. The X-ray crystallographic
structures of E180K in the absence of magnesium and in the presence a
nd 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-ty
pe and both E180K structures show no significant structural difference
s, except the epsilon-amino group of Lys180, which occupies the positi
on usually occupied by the Mg-l. Other active-site residues usually bo
und to Mg-1 are only slightly changed in position, with a magnesium io
n occupying the second metal-binding site (site 2, Mg-2). Structurally
, the lysine residue has successfully replaced Mg-1. Europium(III) exc
itation spectroscopy shows that when Eu3+ is bound to E180K at site 2
(in the absence of substrate), one to two water molecules is coordinat
ed to Eu3+. The X-ray crystallographic structure of the E180K enzyme s
hows that one hydroxide molecule is coordinated to Mg-2. This result i
s consistent with the apparent pK(a) of 7.2 from a plot of pH versus l
og V-max/K-m for the wild-type enzyme. Hydroxide coordinated to Mg-2 c
ould act to deprotonate the glucose O2 and protonate O1 in a step conc
omitant with the hydride-transfer step of isomerization.