Sensitivity of Escherichia coli succinyl-CoA mutants at Trp beta 76 to clostripain and to trypsin. ADP and ATP protect against cleavage by clostripain at Arg beta 80.
Mutant forms of Escherichia coli succinyl-CoA synthetase, W76F (Trp beta 76 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 microM, respectively. Polyacrylamide gel electrophoresis of clostripain digests revealed preferential digestion of the beta-subunit and the appearance of 40- and 31-kDa species, with amino termini corresponding to residues 15 and 81, respectively, of the beta-subunit. Mutagenic replacement of Arg beta 80, but not Arg beta 14, with Lys resulted in an enzyme that was as resistant to clostripain as wild-type enzyme. These results suggest that Arg beta 80 is the principal site of inactivation by clostripain and may be involved in the binding of ADP and ATP to succinyl-CoA synthetase.
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
J S
JS
Nishimura
J
J
Ybarra
C J
CJ
Mann
T
T
Mitchell
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D013389:Succinate-CoA Ligases / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology; D014364:Tryptophan / Q000187:drug effects / Q000378:metabolism*
D000214:Acyl Coenzyme A; D009838:Oligodeoxyribonucleotides; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D001120:Arginine; C012046:succinyl-coenzyme A; D014357:Trypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D013389:Succinate-CoA Ligases