Inactivation of ethanolamine ammonia-lyase by 5,5′-dithiobis(2-nitrobenzoic acid): Further evidence for the involvement of sulfhydryl groups in adenosylcobalamin-dependent rearrangements

Abstract

Treatment of the adenosylcobalamin-requiring enzyme ethanolamine ammonia-lyase (EC 4.3.1.7) with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs 2) discloses three classes of -SH groups: a rapidly reacting class (2 -SH groups/enzyme molecule), a slowly reacting class (6 -SH groups/molecule) and a class which does not react unless the enzyme is denatured (7 -SH groups/molecule (Kaplan, B.H. and Stadtman, E.R. (1968) J. Biol. Chem. 243, 1794)). The enzyme is inactivated by Nbs 2 at a rate similar to the rate at which Nbs 2 reacts with the slowly reacting class of -SH groups. Inactivation of the enzyme is retarded by adenosylcobalamin but not by ethanolamine. Once inactivated, the enzyme cannot be reactivated with mercaptoethanol. These observations provide further evidence for the importance of -SH groups in catalysis by adenosylcobalamin-requiring enzymes.