Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase.

摘要: 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 enzyme activity correlates with incorporation 2 eq N-[ethyl-2-3H]maleimide/subunit. Preincubation methionine ATP analog adenylylimidodiphosphate reduced rate more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified modified reverse phase high performance liquid chromatography. The residues identified as cysteine 90 240 comparison amino acid compositions these protein sequence. These are first to be implicated in and/or structure enzyme. N-Ethylmaleimide-modified S-adenosylmethionine exists mainly a dimer conditions where native tetramer. Accumulation parallels activity. When an sample was partially inactivated, separation tetrameric dimeric forms gel filtration revealed that residual solely present tetramer N-[ethyl-2-3H] maleimide predominantly dimer. Gel studies tetramer-dimer equilibrium for indicated dissociation constant between dimers less 6 x 10(-11) M. Similar N-ethylmaleimide-modified approximately 4 10(-4) Upon modification strength dimer-dimer interactions diminished at least 9 kcal/mol.