Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant b aculovirus-infected insect cells (Yamada, K,, Tohimatsu, T,, and Toraya, T, (1998) Biosci, Biotech, Biochem, 62, 2155-2160). The apoenzyme produced wa s very unstable, and therefore, after complexation with methylcobalamin, th e functional holoenzyme was purified to homogeneity, The specific activity and apparent K-m values for substrates were in good agreement with those ob tained with purified rat liver enzyme. The electronic spectrum of the purif ied recombinant enzyme resembled that of cob(II)alamin and changed to a met hylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine, The rate of oxidative inactivation of the enzyme in t he absence of S-adenosylmethionine was slower with a stronger reducing agen t like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme t o apoprotein and thus for catalysis, Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effectiv e or were effective only slightly in reconstituting holoenzyme, Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were conver ted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by ph otoirradiation.