| It has been observed that thermophilic enzymes are often more sluggish at lower temperatures but comparable active as their mesophilic homologues at their corresponding living temperatures. Although these thermophilic enzymes exhibit high structural stability, the increased stability leads to a decreased flexibility of the thermophilic enzymes in return. To yield further advances in analysis of the interrelationships between flexibility and activity of enzymes, also the molecular basis of enzyme adaptation, we used a pair of thermo-meso acylphosphatase homologues with high level of similarity isolated from hyperthermophilic archeaon Pyrococcus horikoshii (PhAcP) and human (HuAcP) as model to study this issue. Despite the fact that their active-site residues are highly conserved, activity (kcat) of PhAcP is remarkably reduced compared with HuAcP at low temperatures. Based on crystal structure comparison, an extra salt bridge was formed between active site residue and C-terminus of PhAcP. To examine the role of salt bridge plays in catalytic reaction of AcPs, we designed a mutant PhG91A to disrupt the salt bridge in thermophilic PhAcP. In parallel, a salt bridge was re-engineered into mesophilic HuAcP to create HuA99K. Interestingly, the thermophilic variant PhG91A exhibited a more mesophilic-like manner in terms of activity and thermodynamic parameters. On the contrary, mesophilic HuA99K displayed a more thermophilic-like character. This is supplemented by detailed molecular dynamics (MD) simulations, revealing good qualitative agreement with experimental findings. Both theory and experiment results had provided evidences that the presence of a specific salt bridge is directly associated with the temperature adaptation of AcPs by reducing the catalytic site flexibility. |
