A dehydroalanyl residue can capture the 5 '-deoxyadenosyl radical generated from S-adenosylmethionine by pyruvate formate-lyase-activating enzyme

The glycyl radical (Gly-734) contained in the active form of pyruvate forma te-lyase (PFL) of Escherichia coli is produced post-translationally by pyru vate formate-lyase-activating enzyme (PFL activase), employing adenosylmeth ionine (AdoMet) and dihydroflavodoxin as co-substrates. Previous H-2-labeli ngs found incorporation of the pro-S hydrogen of Gly-734 into the 5'-deoxya denosine co-product, indicating that a deoxyadenosyl radical intermediate, generated by reductive cleavage of AdoMet, serves as the actual H atom abst racting species in this system. We have now examined an octapeptide (Suc-Ar g-Val-Pro-Delta Ala-Tyr-Ala-Val-Arg-NH2) that is analogous to the Gly-734 s ite of the PFL polypeptide but contains a dehydroalanyl residue (Delta Ala) in the glycyl position. Applied to the PFL activase reaction, this peptide becomes C-adenosylated at the olefinic beta carbon of Delta Ala. The modif ied peptide was isolated in mu mol-quantities and characterized, after chym otryptic truncation, by MS and 2D NMR. PFL activase functions catalytically (k(cat) greater than or equal to 1 min(-1)) in the peptide modification re action, which occurs with stoichiometric consumption of AdoMet. The mechani sm appears to involve addition of the nucleophilic deoxyadenosyl radical to the electrophilic CC double bond of Delta Ala, followed by quenching of th e peptide backbone-centered adduct radical by the buffer medium. The trappi ng-property of the Delta Ala residue should be exploitable in investigating of how the Fe4S4 protein PFL activase generates the highly reactive deoxya denosyl radical. (C) 1999 Academic Press.