K. Yamada et al., Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase, J BIOL CHEM, 274(50), 1999, pp. 35571-35576
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.