Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase.
S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.
Markham
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GD
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Satishchandran
C
C
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000494:pharmacology; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors*; D010710:Phosphates / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000032:analysis; D014166:Transferases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D046911:Macromolecular Substances; D010710:Phosphates; D013438:Sulfhydryl Compounds; D000266:Adenylyl Imidodiphosphate; D008715:Methionine; D014166:Transferases; D008716:Methionine Adenosyltransferase; D005033:Ethylmaleimide