X-RAY CRYSTALLOGRAPHIC STRUCTURES OF D-XYLOSE ISOMERASE-SUBSTRATE COMPLEXES POSITION THE SUBSTRATE AND PROVIDE EVIDENCE FOR METAL MOVEMENT DURING CATALYSIS

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.