Biosynthesis of the Peptide Bond in the CoenzymeN-(7-Mercaptoheptanoyl)-l-threonine Phosphate

Abstract

The biochemical mechanism for the formation of the amide bond inN-(7-mercaptoheptanoyl)-l-threonine phosphate (HS-HTP) has been studied by measuring the incorporation ofl-[3-3H]threonine intoN-(7-mercaptoheptanoyl)-l-threonine (HS-HT) by cell extracts (CE) ofMethanosarcina thermophilaincubated with different precursors. Synthesis of HS-HT was observed froml-[3-3H]threonine and 7-mercaptoheptanoic acid (HS-H) when the incubations were conducted with either crude CE or Sephadex column-purified CE. In the presence of CE, the synthesis of HS-HT was found to be inhibited 66% by preincubation of the extract with ATPase, indicating that ATP was involved in the biosynthesis. In spite of this indication of ATP involvement in the coupling reaction, incubation of the crude CE withl-[3-3H]threonine, HS-H, and ATP was found to inhibit the formation of HS-HT. In contrast, the synthesis of HS-HT in the presence of Sephadex column-purified CE was found to be stimulated by the addition of ATP. Incubation of the crude CE with the CoA thioester of 7-mercaptoheptanoic acid (HS-HCoA) or the mixed disulfide formed between coenzyme M and 7-mercaptoheptanoic acid did not stimulate the biosynthesis. The biosynthesis of HS-HT was found to be strongly inhibited by an ethanol extract of the crude CE. This inhibition was found to be attributed to the HS-HTP present in the extract. Stimulation of HS-HT biosynthesis 300-fold was observed when the Sephadex column-purified CE was incubated withl-[3-3H]threonine and 7-mercaptoheptanoyl phosphate (HS-H-P). Data indicate that HS-HT is produced by the phosphorylation of HS-H to HS-H-P with ATP, which then reacts withl-threonine to produce HS-HT.