Identification of different classes of nonessential sulfhydryl groups in Escherichia coli adenylosuccinate synthetase.
Reaction of Escherichia coli adenylosuccinate synthetase with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM) leads to modification of one cysteine residue per enzyme monomer without significant loss of enzyme activity. Modification of a second cysteine residue occurs under mild denaturing conditions (3.5 M urea), and derivatization of this thiol followed by dialysis results in complete loss of enzyme activity. The remaining two cysteine residues react with DTNB only after treatment with 8 M urea. The location of the various cysteine residues in the enzyme was established by using [14C]NEM followed by tryptic digestion and radiopeptide isolation. The reactive cysteine has been identified as Cys291, and the thiol exposed with 3.5 M urea is Cys344. When Cys344 was replaced by either serine or alanine, the mutant enzymes were found to be as active as the wild-type enzyme. These findings point to the nonessential role of Cys344 in adenylosuccinate synthetase.
Dong
Q
Q
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Soans
C
C
Liu
F
F
Fromm
H J
HJ
D000265:Adenylosuccinate Synthase / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin
D009579:Nitrobenzoates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D008025:Ligases; D000265:Adenylosuccinate Synthase; D003545:Cysteine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide