Leveraging mechanism-based inhibition to guide rational engineering of enzymatic activity towards polyurethane

Polyurethane (PU) is widely used due to its low-cost production, stability and structural versatility. However, due to inadequate waste management, the widespread use of this material has raised environmental concerns. Recently, academic and industrial efforts have been made to discover enzymes capable of depolymerizing PU. Nevertheless, the enzymes discovered to date lack sufficient efficiency and stability for industrial implementation, requiring further engineering. In this work, we present a new mechanism-based inhibitor (also called a "suicide inhibitor") based on a model substrate representative of industrial PU formulations and designed to bind the enzyme covalently, forming a stable complex that mimics the rate-limiting transition state (TS) for hydrolysis. This is a key structure for rational enzyme optimization which is difficult to observe experimentally, due to its very short lifetime. Therefore, the inhibitor can aid in overcoming this hurdle by forming an experimentally observable complex. We used computational methodologies to model the enzyme:inhibitor complex and study its mechanism of action. Pseudomonas sp. MIS38 lipase was chosen as the template enzyme because thermochemical data is available for its catalytic hydrolysis of the model substrate. The action mechanism of the suicide inhibitor has a low energetic barrier of 9.29 kcal mol^-1 (≈ two-fold lower than that obtained for the substrate) and corresponds to the collapse of the intermediate and elimination of the leaving group. Thus, the designed inhibitor forms a stable covalent bond rapidly. In the enzyme-inhibitor complex, the enzyme nucleophile (Ser207) is bound to the inhibitor's P atom. Moreover, it resembles the rate-limiting TS for urethane bond hydrolysis, as determined by earlier QM/MM calculations. Consequently, this inhibitor can guide rational engineering efforts, by enabling researchers to identify potential mutational targets to enhance enzyme efficiency (increasing the probability of industrial implementation). Altogether, we believe that this inhibitor can be a valuable tool for enabling future engineering efforts.