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 baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.
Yamada
K
K
Department of Bioscience, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan.
Yamada
S
S
Tobimatsu
T
T
Toraya
T
T
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism
D011994:Recombinant Proteins; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12