Catalytic and regulatory sites in CF1.
The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.
Iraburu
M J
MJ
Departamento de Bioquímica Universidad de Navarra, Pamplona, Spain.
López-Zabalza
M J
MJ
Santiago
E
E
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000495:Allosteric Site; D000838:Anions / Q000494:pharmacology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D000838:Anions; D010940:Plant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate