Use of Ammonia-Substituted Chromium Nucleotides for Micromapping of Enzyme Active Sites

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The purpose of this project is to tell which portion of the metal ion coordination sphere faces the enzyme, and which faces the solvent when MgATP or MgADP combine with an enzyme such as hexokinase. To permit such micromapping the investigators will prepare and characterize inert chromium complexes of ATP and ADP which contain one or two ammonias in the coordination sphere. Because ammonia in the coordination sphere lowers affinity when the ammonia is in contact with the protein, but should have little effect when it faces the solvent, the dissociation constants of these complexes will tell which portion of the metal ion coordination sphere contacts the enzyme, and which faces the solvent. The complexes to be characterized are: 1) the 12 isomers of monoamminechromium ATP and the expected corresponding isomers containing ADP. 2) the apparent 10 isomers of cis- diamminechromium ATP, and the corresponding ADP isomers,3) the two expected isomers of trans-diamminechromium ATP or ADP. As an aid to characterizing these isomers, they will try to crystallize the two isomers of monoamminechromium pyrophosphate,and prepare the corresponding tripolyphosphate complexes. They will also try to crystallize one of the enantiomers of cis-diamminechromium pyrophosphate (where water as well as ammonia is cis) by use of a chiral anion or cation, or a chiral solvent. Knowledge of the signs of the CD spectra of these isomers will help characterize the cis-diamminechromium nucleotide complexes. These chromium complexes, once characterized. will be used to map the active sites of hexokinase, pyruvate kinase, adenylate kinase, and phosphofructokinase, all of which accept tetraaquochromium ATP as a substrate.