Co-localization and confinement of ecto-nucleotidases modulate extracellular adenosine nucleotide pools

Abstract

1Nucleotides comprise small molecules that perform critical signaling and energetic roles in biological systems. Of these, the concentrations of adenosine and its derivatives, including adenosine tri-, di-, and mono-phosphate are dynamically controlled in the extracellular-space by diphosphohydrolases and ecto-nucleotidases that rapidly degrade such nucleotides. In many instances, the close coupling between cells such as those in synaptic junctions yields tiny extracellular nanodomains, within which the charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of sequentially-coupled diphosphohydrolase/nucleotidase-catalyzed reactions. To rank the relative importance of these factors, we utilize reaction-diffusion numerical simulations to systematically probe coupled enzyme activity in narrow junctions. We perform these simulations in nanoscale geometries representative of narrow extracellular compartments, within which we localize sequentially- and spatially-coupled enzymes. These enzymes catalyze the conversion of a representative charged substrate such as adenosine triphosphate (ATP) into substrates with different net charges, such as adenosine monophosphate (AMP) and adenosine (Ado). Our modeling approach considers electrostatic interactions of diffusing, charged substrates with extracellular membranes, and coupled enzymes. With this model, we find that 1) Reaction rates exhibited confinement effects, namely reduced reaction rates relative to bulk, that were most pronounced when the enzyme was close to the pore size and 2) The presence of charge on the pore boundary further tunes reaction rates by controlling the pooling of substrate near the reactive protein akin to ions near trans-membrane proteins. These findings suggest how remarkable reaction efficiencies of coupled enzymatic processes can be supported in charged and spatially-confined volumes of extracellular spaces.