NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities. NMR spectroscopy and chemical cross-linking combined with high-resolution mass spectrometry were used to establish that PK binds to ribosome at three independent sites, the L1 stalk, the A site, and the mRNA entry pore. The bioanalytical methodology described characterizes the altered kinetics and confirms the specificity of pyruvate kinase−ribosome interaction, affording an opportunity to investigate the ribosome dependence of metabolic reactions under solution conditions that closely mimic the cytosol. Expanding on the concept of ribosomal heterogeneity, which describes variations in ribosomal constituents that contribute to the specificity of cellular processes, this work firmly establishes the reciprocal process by which ribosome-dependent quinary interactions affect metabolic activity.

Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured cells.

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