Mutant forms of Escherichia coli succinyl-CoA synthetase, W76F (Trp(β76) replaced by Phe) (Nishimura, J. S., Mann, C. J., Ybarra, J., Mitchell, T., and Horowitz, P. M. (1990) Biochemistry 29, 862-865), and W43,76,248F (all three Trp replaced by Phe) were found to be more sensitive to proteolysis by clostripain than the wild-type enzyme or other Trp mutant proteins. Like wild-type enzyme, sensitivity to trypsin was apparent when the enzyme forms were in the dephosphorylated state. Sensitivity to clostripain was the same, whether mutant or wild-type forms were in the phosphorylated or dephosphorylated state. The substrates ADP and ATP both protected the enzymes against inactivation by clostripain, with dissociation constants for protection of W76F of 33 and 125 μM, respectively. Polyacrylamide gel electrophoresis of clostripain digests revealed preferential digestion of the β-subunit and the appearance of 40- and 31-kDa species, with amino termini corresponding to residues 15 and 81, respectively, of the β-subunit. Mutagenic replacement of Arg(β80), but not Arg(β14), with Lys resulted in an enzyme that was as resistant to clostripain as wild-type enzyme. These results suggest that Arg(β80) is the principal site of inactivation by clostripain and may be involved in the binding of ADP and ATP to succinyl- CoA synthetase.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology