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

Abstract

1 1 Abstract 1Nucleotides comprise small molecules that perform critical signaling and en-2 ergetic roles in biological systems. Of these, the concentrations of adenosine 3 and its derivatives, including adenosine tri-, di-, and mono-phosphate are dy-4 namically controlled in the extracellular-space by diphosphohydrolases and ecto-5 nucleotidases that rapidly degrade such nucleotides. In many instances, the close 6 coupling between cells such as those in synaptic junctions yields tiny extracellu-7 lar 'nanodomains', within which the charged nucleotides interact with densely-8 packed membranes and biomolecules. While the contributions of electrostatic 9 and steric interactions within such nanodomains are known to shape diffusion-10 limited reaction rates, less is understood about how these factors control the 11 kinetics of sequentially-coupled diphosphohydrolase/nucleotidase-catalyzed re-12 actions. To rank the relative importance of these factors, we utilize reaction-13 diffusion numerical simulations to systematically probe coupled enzyme activ-14 ity in narrow junctions. We perform these simulations in nanoscale geometries 15 representative of narrow extracellular compartments, within which we localize 16 sequentially-and spatially-coupled enzymes. These enzymes catalyze the con-17 version of a representative charged substrate such as adenosine triphosphate 18 (ATP) into substrates with different net charges, such as adenosine monophos-19 phate (AMP) and adenosine (Ado). Our modeling approach considers elec-20 trostatic interactions of diffusing, charged substrates with extracellular mem-21 branes, and coupled enzymes. With this model, we find that 1) Reaction rates 22 exhibited confinement effects, namely reduced reaction rates relative to bulk, 23 that were most pronounced when the enzyme was close to the pore size and 24 2) The presence of charge on the pore boundary further tunes reaction rates 25 by controlling the pooling of substrate near the reactive protein akin to ions 26 near trans-membrane proteins. These findings suggest how remarkable reaction 27 efficiencies of coupled enzymatic processes can be supported in charged and 28 spatially-confined volumes of extracellular spaces. 29 2 Introduction 30 Nucleotide signaling and regulation of cellular energy pools are reliant on the 31 diffusion of small molecules over micrometer-scale distances [1]. Examples of 32 processes reliant on nucleotides include signal transduction and regulation in 33 smooth muscle [2], network motifs in transcriptional regulation networks [3], 34 genomic regulatory networks [4], complexes of metabolic enzymes [5] and trans-35membrane ligand-gated channels [6, 7]. Of the latter, many nucleotide-gated 36 channels and ATPases [8] reside within extracellular junctions formed between 37 cells in close apposition [9], such as synaptic junctions comprised of neurons 38 and glia [10]. Scanning electron microscopy has revealed that many of these 39 junctions are on the nanometer length-scale [11]. Within those spaces, we ex-4...