Effect of Surface Crowding and Surface Hydrophilicity on the Activity, Stability and Molecular Orientation of a Covalently Tethered Enzyme.

We have investigated two surface properties that are generally thought to have an important influence of enzyme activity and stability: surface hydrophobicity and surface crowding. Here two variants of an engineered bacterial nitro-reductase were covalently tethered to orient the protein's pseudo-2-fold symmetry axis either parallel or perpendicular to the surface. The surface hydrophobicity was systematically varied by changing the ratio of methyl- to hydroxyl-groups displayed on the SAM surface, and the effects on enzyme activity, thermal stability, and structure investigated. Increasing surface hydrophobicity progressively decreased enzyme activity, but had no effect on thermal stability. Surface-sensitive sum frequency generation and attenuated total reflectance Fourier transform IR spectroscopies indicated that the enzyme is not denatured by the more hydrophobic surface, but is more likely trapped in less active conformations by transient hydrophobic interactions. In contrast, increasing enzyme surface concentration increased the specific activity of the parallel oriented enzyme, but had no effect on the activity of the perpendicularly oriented enzyme, suggesting that crowding effects are highly context dependent.