Characterization of isomers of monoamminechromium-ATP and their use in mapping enzyme active sites

Twelve isomers formed by the reaction of monoamminechromium(III) with ATP have been synthesized. Isomerism in this system results from chirality around the beta-phosphorus of the ATP, the position of the ammonia ligand, the relative orientation of the ammonia and the AMP, and the presence of ring-puckering conformers. By using chromatography on cross-linked cycloheptaamylose, reverse-phase C-18 HPLC, and cation-exchange FPLC, these isomers have been separated and purified. Their structures have been identified by (1) cleavage by periodate, followed by elimination in the presence of diethylenetriamine and subsequent phosphate insertion to give lambda, delta, or meso facial monoamminechromium tripolyphosphate with molar ellipticities of +240, -240, or 0 deg cm2 dmol-1 at 550 nm, respectively, (2) cleavage by nucleotide pyrophosphatase to give meridional or facial monoamminechromium pyrophosphate, (3) spectral data, and (4) rates of interconversion of isomers. All possible isomers are seen except those with ammonia syn to AMP. Since the substitution of ammonia for water in the inner coordination sphere appears to diminish affinity for enzymes when the ammonia is in contact with the protein but not when it faces the solvent, these isomers are useful for mapping of enzyme active sites. Their use as probes of enzyme structure is illustrated by their behavior with yeast hexokinase.