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.
Highlights
Metabolic repercussions of methionine synthase dysfunction include depletion of AdoMet and methionine pools with concomitant elevation of homocysteine, a suspected cardiovascular risk factor (Refs. 1 and 2 and references therein)
Resolution of the relative levels ofholo- versus apoenzyme in the cell is complicated by the mixed oxidation states of cobalamin in the isolated methionine synthase and the need for a relatively low potential single electron donor to activate the protein, the estimates of the proportion of holoenzyme have been conflicting, ranging from 10% [5, 6] to 100% [7]
Three are variations of holoenzyme, in which the bound cobalamin is either in an "active form" (IV, CH 3-cobalamin) or in an "inactive form" (II and in an inactive form (III)). The latter two species can be readmitted to the catalytic cycle in a reaction that is independent of added cobalamin but dependent on a reducing system and AdoMet
Summary
Vol 270, No 33, Issue of August 18, pp. 19246-19249, 1995 Printed in U.S.A. Demonstration That Mammalian Methionine Synthases Are Predominantly Cobalamin-Ioaded*. 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. The problem with the standard assay that is routinely employed to monitor methionine synthase activity is that it relies on the supply of exogenous cobalamin (usually in the form of OH-cob(III)alamin) and a reductant such as dithiothreitol, Hydroxocobalamin potentially plays a dual role, It is required in delivering one-electron equivalent to forms II and III, and it can bind to the apoenzyme form (I), converting it to holoenzyme. The cofactor is tightly bound and is retained during multiple purification steps
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