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N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme ofMr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure ofthe enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel ,8-barrel with a-helices on the outside connecting the f8-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the ,8-sheet strands and the subsequent a-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared. MATERIALS AND METHODS Crystals of PRAI-IGPS were obtained as described (14,15). They belong to space group P41 with unit cell parameters a = b = 104.7 A, c = 68.0 A, and one molecule per asymmetric unit (67% solvent content). Screening of heavy atom derivatives was done by soaking crystals in a buffered solution of the appropriate heavy atom compound in 3 M ammonium sulfate (Table 1). For potentially useful derivatives, a low-resolution diffractometer data set was collected, while oscillation data sets to 2.5-A resolution were collected for the three best derivatives. One derivative was prepared by diffusing an iodinated form of the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate (rCdRP) into the crystals. 5-Iodo-rCdRP was prepared as described for rCdRP (16) except that 5-iodoanthranilate (Fluka) was used in place of anthranilate as starting material and was dissolved in dioxane instead of in 50% aqueous ethanol.The crystals were mounted in thin-walled glass capillaries. Diffractometer data were collected as described elsewhere (17) with the following modifications: after crystal alignment, a single test reflection was used to determine the direction of the c-axis of the crystal; several crystals were required per data set due to strong radiation sensitivity; a maximum intensity loss of about 30% was accepted; and intensities were measured by c-scan over 0.8-1.5°, depending on mosaic spread.Oscillation data were collected and processed as described elsewhere (18) with the following modifications: a cooling device kept the crystal at about 40C; data were collected over 900; two sheets of film per film pack were used; crystal orientation was checked by still photographs at the beginning and end of data collection; and the films were digitized, using a 3.0 OD scale.The oscillation and diffractometer data sets (Table 1) were not merged at this point. Heavy atom sites were accepted only if found with both sets of data. They were located by difference Patterson and difference Fourier techniqu...

Aspartate aminotransferase undergoes major shifts in the conformational equilibrium of the protein matrix during transamination. The present study defines the two conformational states of the enzyme by crystallographic analysis, examines the conditions under which the enzyme crystallizes in each of these conformations, and correlates these conditions with the conformational behaviour of the enzyme in solution, as monitored by a fluorescent reporter group.Cocrystallization of chicken mitochondrial aspartate aminotransferase with inhibitors and covalent coenzymesubstrate adducts yields three different crystal forms. Unliganded enzyme forms triclinic crystals of the open conformation, the structure of which has been solved (space group P1) [Ford, G. C., Eichele, G. & Jansonius, J. N. (1980) Proc. Natl Acad. Sci. USA 77, 2559-2563; Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984) J. Mol. Biol. 174,. Complexes of the enzyme with dicarboxylate ligands form monoclinic or orthorhombic crystals of the closed conformation. The results of structure determinations of the latter two crystal forms at 0.44 nm resolution are described here. In the closed conformation, the small domain has undergone a rigid-body rotation of 12 -14" which closes the active-site pocket.Shifts in the conformational equilibrium of aspartate aminotransferase in solution, as induced by substrates, substrate analogues and specific dicarboxylic inhibitors, can be monitored by changes in the relative fluorescence yield of the enzyme labelled at Cysl66 with monobromotrimethylammoniobimane. The pyridoxal and pyridoxamine forms of the labelled enzyme show the same fluorescence properties, whereas in the apoenzyme the fluorescence intensity is reduced by 30%. All active-site ligands, if added to the labelled pyridoxal enzyme at saturating concentrations, cause a decrease in the fluorescence intensity by 40 -70% and a blue shift of maximally 5 nm. Comparison of the fluorescence properties of the enzyme in various functional states with the crystallographic data shows that both techniques probe the same conformational equilibrium.The conformational change that closes the active site seems to be ligand-induced in the reaction of the pyridoxal form of the enzyme and syncatalytic in the reverse reaction with the pyridoxamine enzyme.