The reaction mechanism and substrate specificity of soluble chloroplast coupling factor 1 (CF,) from spinach were determined by using the purified isomers of chromiumnucleotide complexes either as substrates for the enzyme or as inhibitors of the Ca2+-dependent ATPase activity. The isolation of CrADP([32P]Pi) formed upon the addition of the enzyme to [32P]Pi and -bidentate CrADP and the observation that the -bidentate CrADP epimer was 20-fold more effective in inhibiting the Ca2+-dependent ATPase activity than was the epimer suggest that the substrate of phosphorylation catalyzed by CF, is the A-bidentate metal ADP epimer. Tridentate CrATP was hydrolyzed by soluble CF, to CrADP(P!) at an initial rate of 3.2µmol (mg of CF,)-1 min-1, indicating that the tridentate metal ATP is the substrate for ATP hydrolysis. From these results a mechanism for the phosphorylation of ADP catalyzed by coupling factor 1 is proposed whereby the bidentate metal ADP isomer associates with the enzyme, phosphate inserts into the coordination sphere of the metal, and the oxygen of the 0-phosphate of ADP attacks the inorganic phosphate by an SN2 type reaction. The resulting product is the tridentate ATP ligand.
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