title: Interactions between nucleotide binding sites on chloroplast coupling factor during ATP hydrolysis.
abstract: The initial hydrolysis of CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rat constant for the first-order process is independent of substrate concentration but increases hyperbolically to a limiting value of 0.43 s-1 with increasing concentrations of free Ca2+. A mechanism involving a Ca2+-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approximately 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s-1 as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changed. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases