The role of cysteine oxidation in the thermal inactivation of T4 lysozyme.
Wild-type T4 lysozyme contains unpaired cysteine residues at positions 54 and 97. To investigate the role these residues play in the thermal inactivation of the wild-type, we constructed a double mutant with these cysteines replaced with valine and serine. This molecule, T4 lysozyme (C54V/C97S), is more stable than the wild-type to inactivation at 70 degrees C at pH 6.5 and 8.0. Guanidine hydrochloride reactivation experiments and SDS-PAGE on the inactivated products show that the wild-type is susceptible to varying degrees of oxidative damage, depending on buffer conditions, while the cysteine-minus mutant inactivates only by other pathways. The products of thermal, oxidative inactivation of the wild-type are disulfide-linked oligomers. The dependence of inactivation rate on temperature suggests that the formation of these aggregates depends on prior thermal unfolding of the T4 lysozyme molecule.
D000595:Amino Acid Sequence; D003545:Cysteine; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009113:Muramidase / Q000037:antagonists & inhibitors* / Q000235:genetics; D009154:Mutation; D010084:Oxidation-Reduction; D015202:Protein Engineering; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics
D009113:Muramidase; D003545:Cysteine