A High-Throughput Assay Platform for Next-Generation Mechanistic Enzymology and Applications

Abstract

Twentieth-century biochemistry dissected cellular metabolism, identified enzymes responsible for its regulation, and revealed reaction pathways. Nevertheless, enhanced understanding is needed to dissect evolutionary pathways, rationally engineer novel catalysts, and predict disease effects of human alleles. Deep mechanistic studies of active site residues have revealed much about how they contribute to catalysis, but the enzyme scaffold is required for these residues to participate in catalysis. Deep mutational scanning and other current high-throughput approaches have revealed functional effects throughout enzyme scaffolds, but these studies intrinsically lack mechanistic depth. We have developed HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a technology for expression, purification, and assay of >1000 rationally-selected enzyme variants in a single experiment, thereby merging the power of traditional and recent high-throughput technologies. HT-MEK yields quantitative measurements of multiple kinetic and thermodynamic parameters for each variant. As a first demonstration of HT-MEK, we performed enzyme-wide mutational scans of a well-characterized Alkaline Phosphatase superfamily member, PafA. We made thousands of kinetic and thermodynamic measurements for over 1000 PafA variants with a battery of substrates and inhibitors to probe specific catalytic features. Consistent with the simplest expectation, the affecting mutations cluster around the active site regions previously shown to be directly involved in each mechanism. Nevertheless, the extent and direction of these clusters was not predictable, nor was the observation that several clusters extend to the protein surface, demonstrating that HT-MEK can be used to identify surfaces that may allow allosteric control of particular functions. Parallel approaches provide analogous quantitative stability measurements, some of which we will also present. The efficient and systematic dissection of any enzyme or system of enzymes allowed by HT-MEK enables next-generation applications to engineer allostery, novel catalysts, and efficient biosynthetic pathways for use in industry and medicine.