Abstract
The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1, 4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the opposing 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. 270, 14031-14041). In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains. In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules. A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein. A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs. This observation suggests that the amidase activity in the full-length protein is negatively autoregulated. The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage. No metal ion is required for the amidase activity. A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki approximately 10 microM) but does not inhibit the amidase activity.
Highlights
Breviated GSH), are present at high concentrations (0.1–10 mM) in most cells
Preparation of Amidase Fragment and Comparison to Fulllength Protein—Our previous finding that trypsin cleavage of GSP synthetase/amidase after Arg-538 gives a 61.6-kDa Nterminal fragment with only amidase activity suggested that the protein might possess independent amidase and synthetase domains [15], and led us to test whether a smaller N-terminal fragment might independently fold into a functional amidase domain
One with the transposon inserted after nucleotide 675 encoded the smallest N-terminal fragment, amino acids 1–225 of GSP synthetase/amidase fused to the transposon-encoded dipeptide GV
Summary
Materials—Oligonucleotides were purchased from the Harvard Medical School Biological Chemistry and Molecular Pharmacology departmental biopolymer facility or from Integrated DNA Technologies (Coralville, IA). For purification of the synthetase fragment, lysis of cells (17 g of wet cell paste from 6 liters of culture), streptomycin sulfate and ammonium sulfate fractionation steps, and desalting of redissolved ammonium sulfate pellet were carried out as described for the full-length protein [15]. The column was developed with a gradient of NaCl in buffer C (70 ml of 0 –100 mM, 610 ml of 100 –300 mM), and fractions containing synthetase activity were pooled (81 ml eluting at 120 –180 mM NaCl) This solution was made 1.2 M in (NH4)2SO4 by addition of the solid and was chromatographed in 10-ml aliquots on a Phenyl-Superose HR 10/10 column (Pharmacia) equilibrated in 20 mM potassium phosphate, pH 6.8, 5 mM DTT, 1 mM EDTA (buffer D) containing 1 M (NH4)2SO4. SDS-PAGE analysis of the combined fractions showed the fragment to be Ͼ90% pure (Fig. 1, lane 3)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
