Probing stability-activity relationships in the thermophilic proteasome from Thermoplasma acidophilum by random mutagenesis

Structural perturbations (L65H, V12L/M27T, F35V) generated by random mutati on of the beta -subunit were used to probe the relationship between stabili ty and activity in the thermophilic proteasome from Thermoplasma acidophilu m. The optimum temperature for activity of each mutant (approximate to 95 d egreesC) remained unchanged; however, each mutant was significantly less st able than the wild type. Stability, therefore, is not the factor limiting h igh-temperature activity. Interestingly, mutation L65H drastically reduced stability without affecting specific activity over a wide temperature range , providing evidence that activity and stability can be decoupled. To inves tigate the nature of the flexibility introduced by mutation, stability of t he proteasome was examined under pressure. The application of 10,000 psi st abilized the wild-type proteasome 3.4 fold at 97 degreesC. When inactivatio n temperatures were chosen such that the rate of inactivation of the mutant s was similar to that of the wild type, mutants with changes at the intersu bunit interfaces (L65H and V12L/M27T) were similarly stabilized. Pressure w as less effective in stabilizing mutant F35V, however, in which the substit ution may have introduced a new pathway for inactivation.