Probing stability-activity relationships in the thermophilic proteasome from Thermoplasma acidophilum by random mutagenesis.
Structural perturbations (L65H, V12L/M27T, F35V) generated by random mutation of the beta-subunit were used to probe the relationship between stability and activity in the thermophilic proteasome from Thermoplasma acidophilum. The optimum temperature for activity of each mutant (approximately 95 degrees C) remained unchanged; however, each mutant was significantly less stable than the wild type. Stability, therefore, is not the factor limiting high-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 investigate the nature of the flexibility introduced by mutation, stability of the proteasome was examined under pressure. The application of 10,000 psi stabilized the wild-type proteasome 3.4 fold at 97 degrees C. When inactivation temperatures were chosen such that the rate of inactivation of the mutants was similar to that of the wild type, mutants with changes at the intersubunit interfaces (L65H and V12L/M27T) were similarly stabilized. Pressure was less effective in stabilizing mutant F35V, however, in which the substitution may have introduced a new pathway for inactivation.
Beadell
J S
JS
Department of Chemical Engineering, University of California, Berkeley 94720, USA.
Clark
D S
DS
D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011312:Pressure; D046988:Proteasome Endopeptidase Complex; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D021122:Protein Subunits; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex