On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes. Measurements in Escherichia coli.
Escherichia coli cells were transformed with plasmids encoding 12 site-specific variants of T4 lysozyme, and pulse-chase protocols were used to measure the metabolic stability of each protein. The resulting half-lives ranged from 1 h to more than 50 h. Although the metabolic half-lives of T4 lysozymes correlated roughly with their thermal stabilities, three mutant enzymes were clear exceptions. A reasonably temperature-resistant variant, G156D, exhibited a half-life of 1 h. By contrast, two temperature-sensitive variants, T157I and I3G, were as metabolically stable as wild-type T4 lysozyme. Degradation of two short lived variants, L91P and G156P, required ATP both in vivo and in vitro. Degradation of variant and wild-type enzymes was unimpaired in cells lacking the Lon protease, Clp A or Clp P. However, degradation of L91P and G156D was inhibited in Clp B-cells. Decreased proteolysis of L91P was accompanied by its accumulation in inclusion bodies, indicating that Clp B prevents accumulation of aggregated protein either by preventing aggregation of misfolded polypeptides or solubilizing aggregates.
Inoue
I
I
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Rechsteiner
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D002479:Inclusion Bodies; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D000255:Adenosine Triphosphate; D009113:Muramidase; D010450:Endopeptidases