Measuring kcat/KM values for over 200,000 enzymatic substrates with mRNA display

Abstract

Recent advances in enzymology are enabled by the methods for high-throughput kinetic measurements. Common instrumentation-based techniques can examine thousands of enzymatic reactions in parallel, but scaling the throughput further can be challenging. Here, we establish DOMEK (mRNA-display-based one-shot measurement of enzymatic kinetics), an integrated experimental and computational pipeline for ultra-high-throughput kinetic measurements using mRNA display-derived next-generation sequencing data. The method can accurately determine k_cat/K_M specificity constants of post-translational modification enzyme substrates. We benchmark the platform by measuring k_cat/K_M values for ∼2.86 × 10⁵ peptide substrates of a dehydroalanine reductase and leverage the resulting data to build interpretable models of the substrate fitness landscape. The resulting model accurately decomposes reaction activation energies of a peptide substrate into energetic contributions of individual amino acids to reveal microscopic and macroscopic aspects of the enzyme catalysis. Our results establish a generalizable, enzyme-agnostic framework for scaling kinetic measurements to millions of reactions.