PROJECT SUMMARY. Noncanonical amino acids (ncAAs) have myriad valuable applications in the biochemical and biophysical sciences. Their site-specific incorporation into proteins of interest can directly install systematically perturbed residues, sensitive biophysical probes, bio-orthogonal handles, and post-translational modifications (PTMs) at positions of interest. While promising, these applications have been greatly limited by costly materials and labor- intensive, low-yielding preparations. To realize the full potential of ncAAs, I will leverage the recently developed high-throughput microfluidic enzyme kinetics (HT-MEK) platform from the Fordyce and Herschlag laboratories at Stanford University to enable the parallel expression, purification, and quantitative assay of >1,000 ncAA- harboring protein variants on a single microfluidic device. With this approach, it will become feasible and routine to collect >10,000 gold-standard biochemical measurements of ncAA-containing proteins while using less material and effort than is typically required to collect a single such measurement. To illustrate the power and utility of this technique, I will first apply it towards understanding the catalytic mechanisms governing proton transfer at carbon in the model system alanine racemase (AlaR), an important pyridoxal 5’-phosphate (PLP)-dependent enzyme involved in cell-wall biosynthesis. PLP-dependent enzymes account for 4% of all classified enzymatic activities and ~1.5% of prokaryotic reading frames, and they are increasingly important in biotechnology. Although we have a reasonable understanding of how the small- molecule cofactor itself can influence catalysis, the specific contributions of the protein scaffold remain speculative, qualitative, or both. Previous studies that have used traditional site-directed mutagenesis—altering many properties simultaneously—and only examined a handful of variants have failed to deliver a unified view of how this enzyme achieves its c