EVIDENCE FOR A COVALENT INTERMEDIATE IN THE S-ADENOSYL-L-METHIONINE-DEPENDENT TRANSMETHYLATION REACTION CATALYZED BY SIROHAEM SYNTHASE

CysG, also known as uroporphyrinogen III methylase and sirohaem syntha se (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to t ransform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methi onine (AdoMet)-dependent transmethylations, an NAD(+)-dependent dehydr ogenation and a ferrochelation. The apparent tight binding of AdoMet t o this multifunctional enzyme is investigated. The use of a rapid AdoM et binding assay demonstrates that CysG becomes labelled with both [me thyl-H-3]AdoMet and [carboxyl-C-14]AdoMet. Further experiments show th at the CysG-AdoMet complex is subsequently able to methylate uroporphy rinogen III. CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein. A rapid examination of some of the other transmethylases involved in corrin bi osynthesis reveals that they bind the AdoMet in a similar fashion. A m ultistep transmethylation mechanism is proposed to explain the observe d results.