Surface-immobilized enzymes are important for a wide range of technological applications, including industrial catalysis, drug delivery, medical diagnosis and biosensors. However, our understanding of how enzymes and proteins interact with abiological surfaces on the molecular level remains extremely limited. We have compared the structure, activity and thermal stability of two variants of a β-galactosidase attached to a chemically well-defined maleimide-terminated self-assembled monolayer surface through a unique cysteinyl residue. In one case the enzyme is attached through an α-helix, in the other case through an adjacent loop. Both enzymes exhibit similar specific activities and adopt similar orientations with respect to the surface normal, as determined by sum frequency generation and attenuated total reflectance FT-IR spectroscopies. Surprisingly, however, the loop-tethered enzyme exhibits a thermal stability 10 oC lower than the helix-tethered enzyme and 13 oC lower than the enzyme in free solution. Using coarse grain models, molecular dynamics (MD) simulations of the thermal unfolding of the surface-tethered enzymes were able to reproduce these differences in stability. Thus, revealing that tethering through the more flexible loop position provides more opportunity for surface residues on the protein to interact with the surface and undergo surface-induced unfolding. These observations point to the importance of the location of the attachment point in determining the performance of surface-supported biocatalysts and suggest strategies for optimizing their activity and thermal stability through molecular simulations.