PMID: 3754872Jun 15, 1986Paper

Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine.

The Journal of Biological Chemistry
J SumnerR G Matthews

Abstract

Methylenetetrahydrofolate reductase commits tetrahydrofolate-bound one carbon units to use in the regeneration of the methyl group of adenosylmethionine (AdoMet) in eucaryotes and its activity is allosterically inhibited by AdoMet. Limited proteolysis and scanning transmission electron microscopy have been employed to show that the enzyme is a dimer of identical subunits and that each subunit is composed of spatially distinct domains with molecular masses of approximately 40 and 37 kDa (Matthews, R. G., Vanoni, M. A., Hainfeld, J. F., and Wall, J. (1984) J. Biol. Chem. 259, 11647-11650). We now report the use of the photoaffinity label 8-azido-S-adenosylmethionine (8-N3AdoMet) to locate the binding site for the allosteric inhibitor on the 37-kDa domain. In the absence of light, 8-N3AdoMet is itself an inhibitor of methylenetetrahydrofolate reductase activity, with a Ki value 4.8-fold higher than AdoMet, and like AdoMet it induces slow transitions between active and inactive forms. Photoaffinity labeling is dependent on irradiation with ultraviolet light and is prevented by AdoMet but not by ATP. Limited proteolysis of the photolabeled enzyme results in the formation of a labeled 37-kDa fragment which is further processed to a l...Continue Reading

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