The Preparation and Characterization of Cr(III) and Co(III) Complexes of GDP and GTP and Their Interactions with Avian Phosphoenolpyruvate Carboxykinas

The exchange inert coordination complexes, Cr(H 2 O) 4 GDP, Cr(H 2 O) 4 GTP, Cr(NH 3 ) 4 GDP, Cr(NH 3 ) 4 GTP, Co(NH 3 ) 4 GDP, and Co(NH3) 4 GTP have been synthesized and characterized. The Λ and Δ coordination isomers of Cr(H 2 O) 4 GDP, Cr(NH 3 ) 4 GDP, and the four Cr(H 2 O) 4 GTP isomers have been separated by reverse phase HPLC and characterized by their CD spectra. While the isomers of Co(NH 3 ) 4 GTP have not been successfully separated, 31 P NMR spectroscopy reveals the presence of the Λ and Δ forms. The complexes, Cr(H 2 O) 4 GDP, Co(NH 3 ) 4 GDP, Cr(H 2 O) 4 GTP, and Co(NH 3 ) 4 GTP, are linear competitive inhibitors of avian phosphoenolpyruvate carboxykinase. The K i values of 30 μM, 540 μM, 40 μM, and 12 μM, respectively, were determined for these complexes using Mn-IDP as the nucleotide substrate in the phosphoenolpyruvate carboxylation direction or Mn-ITP as nucleotide substrate for the oxalacetate decarboxylation reaction. The Λ and Δ isomers of Cr(H 2 O) 4 GDP show little specificity (a twofold maximum difference in K i ) for the enzyme. The isomeric forms of Cr(H 2 O) 4 GTP demonstrate no observed stereoselectivity of interaction with the enzyme. All of the complexes tested, except for Cr(NH 3 ) 4 GDP and Co(NH 3 ) 4 GDP, which have larger K i values, are good substrate analogs for P -enolpyruvate carboxykinase. When the substrate is Mn-GTP, fixed at 0.2 mM at pH 6.0, enzyme activity is stimulated two- to two and a half-fold by Cr(H 2 O) 4 GTP. A Dixon plot reveals that the stimulatory effect is saturated at 0.4 mM Cr(H 2 O) 4 GTP. The interaction of the enzyme with Cr(H 2 O) 4 GTP appears to produce a “memory” effect which is manifest with guanosine nucleotide substrates, but which is not observed with the alternative substrate Mn-ITP.