Bringing Enzymology into the Genomic Era: Developing and Deploying New Tools to Quantitatively Map Functional Connections Throughout an Enzyme

Abstract

Despite enormous advances in our understanding of enzymes and their mechanisms over the past decades, how they achieve their enormous rate enhancements and exquisite specificities remains elusive, as evidenced by our limited ability to engineer new enzymes. A fundamental reason for this lack of understanding is the large number of interconnected residues and long-range interactions that cannot be revealed via traditional, low-throughput quantitative assays. While high-throughput screens and selections can identify enzymes with desired changes, the improvements obtained are typically modest and the qualitative or limited quantitative data obtainable by these approaches is insufficient to reveal functional interconnections between remote and active site residues. To meet this unmet need, we have developed a new technology, HT-MEK (High-throughput Microfluidic Enzyme Kinetics), that allows measurement of myriad quantitative biochemical parameters for 1000 rationally chosen enzyme variants in a single experiment. Using this platform, we carried out deep functional analysis of multiple substitutions at each position within a single enzyme, the Alkaline Phosphatase superfamily member PafA. For each substitution, we measured multiple biochemical parameters, including Michaelis-Menten constants for cognate and non-cognate substrates and inhibition constants for multiple inhibitors and transition state analogs. The results provide a detailed functional signature for each position within PafA. We observe distinct physically contiguous ‘functional units’ throughout the enzyme that tune specific aspects of function, yielding blueprints for future rational design efforts. HT-MEK is applicable to enzyme function and stability, provides data that rivals or exceeds the precision typically obtained from standard assays, and reduces the time, effort, and cost to quantitatively profile the effects of individual substitutions on enzyme function by orders of magnitude, providing a technology to access previously inaccessible questions across multiple systems and disciplines.