Large Image

Abstract

The structural flexibility of enzymes plays an essential role in determining their catalytic efficiency and thermal stability. Cold-adapted enzymes are typically highly flexible, resulting in high catalytic activity but low stability. Glucokinase (GK) consists of the large substrates binding domain, small catalytic domain, and hinge region that undergoes conformational changes upon substrates binding. We recently reported that the psychrophilic GK from Pseudoalteromonas sp. AS-131 (PsGK) exhibits both high catalytic efficiency and remarkable thermal stability compared to the mesophilic GK from Escherichia coli (EcGK). We also found that a disulfide bond connecting the N- and C-termini in PsGK contributes to its unusual thermal stability. However, cold adaptation mechanism of cold-adapted PsGK has remained unclear. To clarify how PsGK acquires high activity, we utilized site-directed spin labeling electron spin resonance (SDSL-ESR) spectroscopy for PsGK and EcGK in the absence and presence of substrates in the wide range of temperatures. PsGK without substrates was more flexible than EcGK. Particularly, the small domain and hinge region of PsGK were highly flexible while its large domain was relatively rigid. In contrast, EcGK showed lower entire flexibility and did not exhibit domain dependent differences. When the substrates were bound, both enzymes became more rigid, but the small domain and hinge region of PsGK was still flexible whereas its large domain was considerably rigid. These results suggest that enhancing catalytic activity requires increasing flexibility only in proper sites rather than in the entire enzyme. These findings provide insight into how cold-adapted enzymes balance activity and stability.