DISSECTION OF GLUTATHIONYLSPERMIDINE SYNTHETASE AMIDASE FROM ESCHERICHIA-COLI INTO AUTONOMOUSLY FOLDING AND FUNCTIONAL SYNTHETASE AND AMIDASE DOMAINS/

The bifunctional glutathionylspermidine synthetase/amidase from Escher ichia coli catalyzes both the ATP-dependent formation of an amide bond between N-1 of spermidine (N-(3-amino)propyl-1,4-diaminobutane) and t he glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the oppo sing hydrolysis of this amide bond (Bollinger, J. M., Jr., Kwon, D. S. , Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) J. Biol. Chem. 2 70, 14031-14041). In our previous work describing its initial characte rization, we proposed that the 619-amino acid (70 kDa) protein might p ossess separate amidase (N-terminal) and synthetase (C-terminal) domai ns. In the present study, we have confirmed this hypothesis by express ion of independently folding and functional amidase and synthetase mod ules. A fragment containing the C-terminal 431 amino acids (50 kDa) ha s synthetase activity only, with steady-state kinetic parameters simil ar to the full-length protein. A fragment containing the N-terminal 22 5 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutat hionylspermidine analogs. This observation suggests that the amidase a ctivity in the full-length protein is negatively autoregulated, The am idase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) bu t lacks activity toward acetylspermidine (N-1 and N-8) and acetylsperm idine (N-1), indicating that glutathione provides the primary recognit ion determinants for glutathionylspermidine amide bond cleavage. No me tal ion is required for the amidase activity, A tetrahedral phosphonat e analogue of glutathionylspermidine, designed as a mimic of the propo sed tetrahedral intermediate for either reaction, inhibits the synthet ase activity (K-i similar to 10 mu M) but does not inhibit the amidase activity.