Schiano-di-Cola, C. | Search bioRxiv

bioRxiv2024

Interrelationship of substrate crystallinity, enzyme binding strength, and cellulase activity

Structural polysaccharides are difficult to degrade due to their crystalline structure. Hence, industrial conversion of biomass has focused on both substrate pretreatment and enzyme engineering to improve the biochemical conversion of biomass into fuels and chemicals. However, few studies have explored the interrelationship between substrate crystallinity and cellulase activity. Here, we systematically investigated the kinetics of structurally diverse cellulases on five cellulosic substrates with varying crystallinity. Regardless of enzyme structure and catalytic mechanism, we observed a linear scaling of the kinetic parameters (KM and kcat) in a log-log plot, indicating a linear free energy relationship (LFER) between binding and activation energy. LFERs were observed for all investigated substrates, but their slopes varied distinctly and appeared linked to the substrate crystallinity. Substrates with low crystallinity exhibited LFERs with a slope near 1, while highly crystalline substrates had a slope of approximately 0.25, providing insights into the transition state (TS) for the rate-limiting step. We propose that maximal turnover was limited by slow dissociation, with the TS structurally close to the enzyme-ligand complex on crystalline substrate, while on amorphous substrate, the TS structure was closer to the dissociated system. We suggest that these observations reflect competing interactions of the ligand with respectively the enzyme binding cleft and the substrate matrix. This study emphasizes the interconnected nature of substrate pretreatment and enzyme engineering, urging a holistic approach to propel the biochemical conversion of lignocellulosic biomass, crucial for advancing sustainable production of fuels and chemicals.

biochemistry↗

bioRxiv2020

Physical constrains and functional plasticity of cellulases: Linear scaling relationships for a heterogeneous enzyme reaction

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.

biochemistry↗