A New Approach to the Mechanism of Photophosphorylation: Modulation of ATP Synthetase Activity by Limited Diffusibility of Nucleotides near the Enzyme

This chapter presents experimental procedures, showing that the steady-state rate of phosphorylation may be limited by the mass transfer of nucleotides between the adenosine triphosphate (ATP) synthetase loci and the bulk medium. The data presented in the chapter indicates the limitation of catalysis by mass transfer of nucleotides through the environment of the membrane in normally prepared thylakoids. Most of the ATP, rapidly labeled in the light and retained on de-energized membranes, is not bound to the catalytic sites of ATP synthetase and probably originates from a free species of newly formed ATP, which diffuses slowly to the bulk medium. Hypotonic treatment and washing of normally prepared thylakoids, which could remove kinetic barriers to diffusion by opening grana stacks, increases the apparent affinity of the membrane-bound ATP synthetase for substrate. This effect seems to depend on the catalytic ability of the thylakoids and their efficiency of substrate utilization. The chapter proposes a model that introduces a new perspective to explain modulation of ATP synthetase activities and medium exchange reactions by energy input, substrate concentration, and, in general, any factor that could affect the microenvironment of the enzyme, provided that proper conditions for diffusion control are present.