Catalysis by isolated beta-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of pyrophosphate.
Although the capacity of isolated beta-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F(1) of mitochondrial H(+)-ATPase can bind inorganic pyrophosphate (PP(i)) and synthesize PP(i) from medium phosphate, we examined if purified His-tagged beta-subunits from Thermophilic bacillus PS3 can hydrolyze PP(i). The difference spectra in the near UV CD of beta-subunits with and without PP(i) show that PP(i) binds to the subunits. Other studies show that beta-subunits hydrolyze [(32)P] PP(i) through a Mg(2+)-dependent process with an optimal pH of 8.3. Free Mg(2+) is required for maximal hydrolytic rates. The Km for PP(i) is 75 microM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP(i). Thus, isolated beta-subunits are catalytically competent with PP(i) as substrate; apparently, the assembly of beta-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.
José-Nuñez
Concepción
C
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Apartado Postal 70243, 04510, México, D. F., México.
Torres-Larios
Alfredo
A
Ramírez-Silva
Leticia
L
Mendoza
Guillermo
G
Salcedo
Guillermo
G
Gómez-Puyou
Armando
A
Tuena de Gómez-Puyou
Marietta
M
D025181:ATP Synthetase Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001105:Archaea / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D006868:Hydrolysis; D012995:Solubility
D001426:Bacterial Proteins; D025181:ATP Synthetase Complexes