The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and Cl of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C' or C2 of the substrate. A water molecule binds to C'O and C20 of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate. We (at the University of Strasbourg, V.B., F.M., D.T., and J.F.B.) designed and synthesized a substrate analog that is a suicide inactivator, analog 2 (Scheme 1) (9). It was used to test whether the D-xylose isomerase studied by x-ray diffraction techniques by us (at the Fox Chase Cancer Center, H.L.C. and J.P.G.) is active in crystalline state. Three crystal structures have been determined and each has been refined: native enzyme, enzyme with bound substrate/product, and MATERIALS AND METHODS Preparation of Active-Site-Directed Inhibitor. 3-Deoxy-C3-methylene-D-glucose (analog 1) and (E)-3-deoxy-C3-fluoromethylene-D-glucose (analog 2) were prepared as follows. Chromic oxidation (10) of the hydroxyl group at C3 of 1,2:5,6-diisopropylidene-a-D-glucose (11) gave the ketone 3. The Wittig reaction of ketone 3 with ylide prepared from triphenylmethylphosphonium bromide with potassium t-butanolate (12) or with the ylide derived from fluoromethyl triphenylphosphonium tetrafluoroborate (13) with n-butyllithium and potassium t-butanolate (14) gave, after quantitative acid hydrolysis (15) of the acetonides (16, 17), the glucose analogs 1 § and 2$ (Scheme 1). Anomers of analogs 1 and 2 were detected in aqueous solutions by NMR spectroscopy.Assay ofXylose Isomerase. The enzyme from S. rubiginosus was assayed by the cysteine-carbazole test (18) as the quantity of D-fructose formed. All reactions were performed in a 0.2 M sodium phosphate buffer (pH 8.0) at 40°C. The reaction mixture (1 ml) contained 0.8 M glucose, 0.01 M tTo whom reprint requests should be addressed. §In a total yield of 80%: m.p. 123-125. 13C-NMR (50 MHz, 2H20) 8 (ppm): 148; 146.4; 98.8; 9...

Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.