Development of a kinetic model and figures of merit for formaldehyde carboligations catalyzed by formolase enzymes.

There is an increasing interest in the upgrading of inexpensive and abundant C feedstocks to higher carbon products. Linear carbon ligation routes are of particular interest due to their simplicity and potential for high carbon efficiencies. The formolase (FLS) enzyme was computationally designed to catalyze the formose reaction, where formaldehyde molecules are coupled to produce a mixture of C (glycolaldehyde) and C (dihydroxyacetone) molecules. Recent protein engineering efforts have resulted in the introduction of several FLS variants with altered catalytic properties. As is often the case with enzymes catalyzing reactions with complex and/or nonnatural trajectories, there are no mechanistic kinetic models that fully describe the activity of the FLS enzyme. FLS variants are typically evaluated by fitting rate data to empirical rate laws, with some variation of the k /K ratio used to report and rank performances. The apparent parameters estimated in this manner are unlikely to capture the full catalytic performance of these enzymes. In this study, we derive a mechanistic rate law describing FLS activity as well as theory-based figures of merit to rank FLS performance under relevant conditions. We proceed to fit the rate equation to initial rate data obtained from several FLS mutants, and use the figures of merit to compare the mutations. This study provides a theoretical framework for comparing FLS enzymes which will be essential as novel carbon ligation pathways are devised and implemented.