Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

Abstract

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration.

Anatomy of enzyme channels

“…Here we focus on these channels, as the active site access paths play an important role in substrate and product trafficking between active site and outside. It has been shown that mutations in enzymes’ active site access channels alter the substrate preferences of haloalkane dehalogenase enzymes and may be utilized in rational design of enzymes [26,27]. The amino acids lining the access channels of cytochrome P450 (CYP) are important for the selectivity of these enzymes [28] while the flexibility of these channels, i.e.…”

Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein

“…Volumes of the FMN binding sites were calculated using an in-house code attached as supplemental material that is based on a procedure described by Stepankova et al [28]. That procedure uses atoms that define the location of the binding site of interest in a snapshot from the MD simulation, and a sphere enclosing the measured space.…”

Ensemble Generation and the Influence of Protein Flexibility on Geometric Tunnel Prediction in Cytochrome P450 Enzymes

“…Recent evidence has highlighted the importance of protein flexibility in protein access and egress tunnels [9], [10]. For instance, Stepankova et.…”

“…al. demonstrated that at low concentrations organic solvents cause the widening of tunnels in haloalkane dehalogenase enzymes resulting in either increased activity or inhibition depending on the system and the solvent used [10]. Furthermore, in several cytochrome P450 (CYP) enzymes, phenylalanine clusters located in ligand entrance tunnels just above the active site act as gates that must be properly oriented to allow substrate binding [9], [11].…”