Catalytic properties of the Escherichia coli proton adenosinetriphosphatase: evidence that nucleotide bound at noncatalytic sites is not involved in regulation of oxidative phosphorylation.
Nucleotide-depleted F1-ATPase from Escherichia coli was reconstituted with F1-depleted membranes and shown to catalyze high rates of oxidative phosphorylation of ADP and GDP. Adenine nucleotide became bound to the nonexchangeable nucleotide sites on membrane-bound F1 during ATP synthesis, but binding of guanine nucleotides to nonexchangeable sites during GTP synthesis was not detectable. It was possible to reload the nonexchangeable sites on nucleotide-depleted F1 with radioactive adenine nucleotide prior to membrane reconstitution. The radioactive adenine nucleotide did not exchange significantly during oxidative phosphorylation of ADP or GDP. The amount of nonexchangeable adenine nucleotide found in membrane-bound F1 was the same when the nonexchangeable sites were reloaded either prior to membrane reconstitution of the F1 or after membrane reconstitution with nucleotide-free F1 followed by a burst of oxidative phosphorylation of ADP. The results showed that occupation of the nonexchangeable sites on F1 by tightly bound nucleotide is not required for oxidative phosphorylation of GDP (a physiological activity of F1 in the bacterial cell). Also, the results confirm directly that the adenine-specific nonexchangeable sites on F1 are noncatalytic sites. Using this experimental approach, it was possible to look for a regulatory effect of the nonexchangeable nucleotide on oxidative phosphorylation. Nucleotide-depleted F1 was first reloaded with (i) ATP, (ii) ADP, (iii) 5'-adenylyl imidodiphosphate, or (iv) zero nucleotide, and was then reconstituted with F1-depleted membranes. The reconstituted membranes were compared in respect to rates of oxidative phosphorylation of GDP and Km values of GDP and Pi. No regulatory role for the nonexchangeable nucleotide was evident.(ABSTRACT TRUNCATED AT 250 WORDS)
Wise
J G
JG
Senior
A E
AE
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D010085:Oxidative Phosphorylation; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; D006153:Guanosine Diphosphate; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D006180:Proton-Translocating ATPases