Biosensor-enabled single-molecule enzyme activity profiling for function-based molecular counting

Single-molecule enzyme activity analysis enables proteoform-resolved quantification of catalytically active enzyme molecules but remains limited by the need for enzyme-specific fluorogenic substrates, particularly for enzymes involved in central metabolism. Here we establish a biosensor-based framework that integrates genetically encoded fluorescent metabolite biosensors with single-molecule enzyme activity profiling (SEAP), enabling native enzymatic reactions to be monitored without enzyme-specific fluorogenic substrates. By employing different biosensors, the framework was applied to distinct metabolic enzymes, including lactate dehydrogenase and pyruvate kinase. Biosensor-based SEAP enables absolute quantification of active enzyme molecules, transforming conventional activity measurements (U/mL) into molecular abundance (molecules/mL). Applying this approach to single-cell functional proteoform analysis revealed functional heterogeneity in lactate dehydrogenase activity during T-cell activation. In addition, analysis of circulating enzymes showed that much of the plasma lactate dehydrogenase pool could be quantitatively accounted for by physiological erythrocyte turnover. These results establish genetically encoded fluorescent protein-based metabolite biosensors as modular optical transducers for single-molecule functional enzyme analysis across cellular and circulating samples.