Demonstration that mammalian methionine synthases are predominantly cobalamin-loaded.
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.
Chen
Z
Z
Biochemistry Department, University of Nebraska, Lincoln 68588-0664, USA.
Chakraborty
S
S
Banerjee
R
R
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004579:Electron Transport; D005260:Female; D006801:Humans; D007700:Kinetics; D008322:Mammals; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013552:Swine; D014805:Vitamin B 12 / Q000032:analysis*
D002951:Citrates; D019343:Citric Acid; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12