δ- l-(α-Aminoadipoyl)- l-cysteinyl- d-valine synthetase: isolation of l-cysteinyl- d-valine, a ‘shunt’ product, and implications for the order of peptide bond formation

Abstract

l-Cysteinyl- d-valine was isolated from incubations of l-glutamate, l-cysteine and l-valine with δ- l-(α-aminoadipoyl)- l-cysteinyl- d-valine synthetase and identified by 1H NMR and electrospray ionization MS. This is entirely consistent with our prior proposal (Shiau, C.-Y., Baldwin, J.E., Byford, M.F., Sobey, W.J. and Schofield, C.J. (1995) FEBS Lett. 358, 97–100) that the α-peptide bond between cysteine and valine is formed before the δ-peptide bond between α-aminoadipate and cysteine. The inclusion of l-glutamate, an analogue of l-α-aminoadipate, did not result in a detectable amount of tripeptide product, but did increase apparent yields of l-cysteinyl- d-valine. Conceivably, formation of the l-glutamyladenylate stimulates synthesis of the cysteinyl-valine dipeptide indirectly via a conformational change in the enzyme.