X-RAY CRYSTALLOGRAPHIC STRUCTURES OF D-XYLOSE ISOMERASE-SUBSTRATE COMPLEXES POSITION THE SUBSTRATE AND PROVIDE EVIDENCE FOR METAL MOVEMENT DURING CATALYSIS
A. Lavie et al., X-RAY CRYSTALLOGRAPHIC STRUCTURES OF D-XYLOSE ISOMERASE-SUBSTRATE COMPLEXES POSITION THE SUBSTRATE AND PROVIDE EVIDENCE FOR METAL MOVEMENT DURING CATALYSIS, Biochemistry, 33(18), 1994, pp. 5469-5480
The X-ray crystallographic structures of the metal-activated enzyme xy
lose isomerase from Streptomyces olivochromogenes with the substrates
D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were d
etermined to 1.96-,2.19-, and 1.81-Angstrom resolution and refined to
R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose isomerase c
atalyzes the interconversion between glucose and fructose (xylose and
xylulose under physiological conditions) by utilizing two metal cofact
ors to promote a hydride shift; the metals are bridged by a glutamate
residue. This puts xylose isomerase in the small but rapidly growing f
amily of enzymes with a bridged bimetallic active site, in which both
metals are involved in the chemical transformation. The substrate 3-O-
methylglucose was chosen in order to position the glucose molecule in
the observed electron density unambiguously. Of the two essential magn
esium ions per active site, Mg-2 was observed to occupy two alternate
positions, separated by 1.8 Angstrom, in the substrate-soaked structur
es. The deduced movement was not observed in the structure without sub
strate present and is attributed to a step following substrate binding
but prior to isomerization. The substrates glucose and 3-O-methylgluc
ose are observed in their linear extended forms and make identical int
eractions with the enzyme by forming ligands to Mg-1 through O2 and O4
and by forming hydrogen bonds with His53 through O5 and Lys182 throug
h O1. Mg-2 has a water ligand that is interpreted in the crystal struc
ture in the absence of substrate as a hydroxide ion and in the presenc
e of substrate as a water molecule. This hydroxide ion may act as a ba
se to deprotonate the glucose O2 and subsequently protonate the produc
t fructose O1 concomitant with hydride transfer. Calculations of the s
olvent-accessible surface of possible dimers, with and without the alp
ha-helical C-terminal domain, suggest that the tetramer is the active
form of this xylose isomerase.