Mechanism of phosphorylation catalyzed by chloroplast coupling factor 1. Stereochemistry.
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 mumol (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 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 beta-phosphate of ADP attacks the inorganic phosphate by an SN2 type reaction. The resulting product is the tridentate ATP ligand.
D000227:Adenine Nucleotides / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008968:Molecular Conformation; D010944:Plants / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D008565:Membrane Proteins; D006180:Proton-Translocating ATPases