The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates (Wagner, A.F. V., Frey, M., Neugebauer, F.A., Schäfer, W., and Knappe, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 996-1000). Studying radical synthesis with [2-2H]glycine-labeled PFL, we have now found stoichiometric incorporation of a 2H atom into the 5'-deoxyadenosine (dAdo) co-product via mass and NMR spectroscopic analyses. Furthermore, a series of peptides homologous to the Gly-734 site of PFL have been synthesized for analyzing recognition determinants of PFL activase. Peptides that proved active as substrates (monitored by [14C]dAdo formation from [14C]AdoMet) were also competitive inhibitors of PFL conversion to the radical form. In the sequence of the standard peptide Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to amino acid residues 731-737 of PFL, the Gly residue was replaceable by D-Ala (actually displaying enhanced efficiency), whereas a normal Ala totally abolished the interaction with PFL activase. Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. Gly-734 is probably located in a beta-turn segment of the protein.