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 chira lity around the beta-phosphorus of the ATP, the position of the ammoni a ligand, the relative orientation of the ammonia and the AMP, and the presence of ring-puckering conformers. By using chromatography on cro ss-linked cycloheptaamylose, reverse-phase C-18 HPLC, and cation-excha nge FPLC, these isomers have been separated and purified. Their struct ures have been identified by (1) cleavage by periodate, followed by el imination in the presence of diethylenetriamine and subsequent phospha te 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 pyrophospha tase to give meridional or facial monoamminechromium pyrophosphate, (3 ) spectral data, and (4) rates of interconversion of isomers. All poss ible isomers are seen except those with ammonia syn to AMP. Since the substitution of ammonia for water in the inner coordination sphere app ears to diminish affinity for enzymes when the ammonia is in contact w ith the protein but not when it faces the solvent, these isomers are u seful for mapping of enzyme active sites. Their use as probes of enzym e structure is illustrated by their behavior with yeast hexokinase.