Demonstration That Mammalian Methionine Synthases Are Predominantly Cobalamin-loaded

Abstract

Methionine synthase is an important cellular housekeeping enzyme and is dependent on the cofactor cobalamin, a derivative of vitamin B12, for activity. It functions in two major metabolic pathways including the tetrahydrofolate-dependent one-carbon cycle and the salvage pathway for methionine. Its dysfunction has several physiological ramifications and leads to the development of megaloblastic anemia. In addition, it is suspected to be involved in the pathogenesis of neural tube defects. An issue that is central in weighing therapeutic options for methionine synthase-related disorders is the extent to which the enzyme exists as apoenzyme in vivo and, thus, can be potentially responsive to vitamin B12 therapy. despite the importance of this issue, the extent of holo- versus apoenzyme in mammalian tissue is controversial and unresolved. To address this question, we have developed a convenient anaerobic assay that employs titanium citrate to deliver low potential electron equivalents. The reductive activation of this enzyme is essential under in vitro assay conditions. We find that both the human placental and porcine liver methionine synthases exist predominantly in the holoenzyme form (90-100%) in the crude homogenate. In addition, the activity of the pure enzyme measured in the titanium citrate assay is also independent of exogenous cofactor, revealing that the cobalamin is tightly bound to the active site.

Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse

“…Recent studies on purified Mtr and Cbs have shown reciprocal sensitivity of these two enzymes to oxidative conditions; Mtr is reduced (Chen et al, 1995) and Cbs is increased (Taoka et al, 1998) under oxidizing conditions. Thus, redox changes may serve a regulatory role such that oxidative stress would increase the flux of methionine through the transsulfuration pathway ultimately increasing the concentration of GSH .…”

Posttranscriptional Regulation of Mammalian Methionine Synthase by B12

“…In the absence of OHB,,, dithiothreitol is unable to transfer electrons to methionine synthase (21). The problem with this assay arises from its dependence on OHB,,, which serves both to transfer electrons from dithiothreitol to activate oxidized holoenzyme and binds to apoenzyme, eventually converting it to holoenzyme (12). Thus, the complexity of the methionine synthase assay has resulted in significant underestimation of the holoenzyme content in the literature.…”

“…This problem is addressed by employing titanium citrate as the electron donor since it can directly reduce methionine synthase (12). Under these assay conditions, the relative content of apo-and holoenzyme can be evaluated by measuring activity in the presence and absence of exogenous OHB,,.…”

Thioredoxin-dependent Enzymatic Activation of Mercaptopyruvate Sulfurtransferase

“…Furthermore, as the sulfenate is at a low redox potential, Trx and DTT reduce it to restore its activity, but reduced glutathione does not affect the activity (13). Under oxidizing conditions, the cysteine pool is increased because of post-translational inhibition of methionine synthase (14,15) and post-translational activation of cystathione ␤-synthase (14,16). Inhibition of MST conserves cysteine and contributes to increase the cysteine pool.…”