Mechanism of phosphorylation catalyzed by chloroplast coupling factor 1. Stereochemistry

Abstract

The reaction mechanism and substrate specificity of soluble chloroplast coupling factor 1 (CF1) from spinach were determined by using the purified isomers of chromium-nucleotide 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 .LAMBDA.-bidentate CrADP and the observation that the .LAMBDA.-bidentate CrADP epimer was 20-fold more effective in inhibiting the Ca2+-dependent ATPase activity than was the .DELTA. epimer suggest that the substrate of phosphorylation catalyzed by CF1 is the .LAMBDA.-bidentate metal ADP epimer. Tridentate CrATP was hydrolyzed by soluble CF1 to CrADP(Pi) at an initial rate of 3.2 .mu.mol (mg of CF1)-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 CF1 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 .beta.-phosphate of ADP attacks Pi by an SN2 type reaction. The resulting product is the tridentate ATP ligand.