Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

Abstract

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V m for ADP-arsenate synthesis was about 2-times lower than V m for ATP-synthesis. With saturating [ADP], K(As i) was about 20% higher than K(P i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of 1 v vs. 1 [ substrate] were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased V m K m ratio. High concentrations of the fixed substrate cause a similar increase in V m K m , but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As i) stimulate ATP synthesis supported by low concentrations of P i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K i of As i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K m of As i for arsenylation; the apparent K i of P i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K m of P i for ATP synthesis. (6) The results are discussed in terms of a model in which P i and As i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.