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

Kazuhiro Yamada, Seiki Yamada, Takamasa Tobimatsu, Tetsuo Toraya

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)35571-35576
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number50
DOIs
Publication statusPublished - Dec 10 1999

Fingerprint

5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase
Apoenzymes
Vitamin B 12
Holoenzymes
Purification
Rats
S-Adenosylmethionine
Reducing Agents
Enzymes
Titanium
Apoproteins
Baculoviridae
Coenzymes
Complexation
Catalysis
Liver
Insects
Nucleotides
Chemical activation
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase. / Yamada, Kazuhiro; Yamada, Seiki; Tobimatsu, Takamasa; Toraya, Tetsuo.

In: Journal of Biological Chemistry, Vol. 274, No. 50, 10.12.1999, p. 35571-35576.

Research output: Contribution to journalArticle

@article{6f6fa0cacf324f8c8dd8827009a76db6,
title = "Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase",
abstract = "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.",
author = "Kazuhiro Yamada and Seiki Yamada and Takamasa Tobimatsu and Tetsuo Toraya",
year = "1999",
month = "12",
day = "10",
doi = "10.1074/jbc.274.50.35571",
language = "English",
volume = "274",
pages = "35571--35576",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "50",

}

TY - JOUR

T1 - Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase

AU - Yamada, Kazuhiro

AU - Yamada, Seiki

AU - Tobimatsu, Takamasa

AU - Toraya, Tetsuo

PY - 1999/12/10

Y1 - 1999/12/10

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0033544939&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033544939&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.50.35571

DO - 10.1074/jbc.274.50.35571

M3 - Article

C2 - 10585432

AN - SCOPUS:0033544939

VL - 274

SP - 35571

EP - 35576

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -