Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.
The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.
Hisabori
T
T
Department of Biology, Yokohama City University, Japan.
Muneyuki
E
E
Odaka
M
M
Yokoyama
K
K
Mochizuki
K
K
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases