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Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.