1108
T. GENDA et al.
(Ki0 value of 12 mM), being two orders larger than that
(Ki ¼ 0:05 mM, competitive) of the pig-heart emzyme.20)
Maleate, the diastereoisomer of fumarate, a competitive
inhibitor for pig-heart fumarase,39) did not inhibit the
C. glutamicum enzyme.
8) Kobayashi, K., and Tuboi, S., End group analysis of the
cytosolic and mitochondrial fumarases from rat liver.
J. Biochem., 94, 707–713 (1983).
9) Woods, S. A., Schwartzbach, S. D., and Guest, J. R.,
Two biochemically distinct classes of fumarase in
Escherichia coli. Biochim. Biophys. Acta, 954, 14–26
(1988).
10) Yumoto, N., and Tokushige, M., Characterization of
multiple fumarase proteins in Escherichia coli. Biochem.
Biophys. Res. Commun., 153, 1236–1243 (1988).
11) Ueda, Y., Yumoto, N., Tokushige, M., Fukui, K., and
Ohya-Nishiguchi, H., Purification and characterization
of two types of fumarase from Escherichia coli. J.
Biochem., 109, 728–733 (1991).
12) Weaver, T. M., Levitt, D. G., and Banaszak, L. J.,
Purification and crystallization of fumarase C from
Escherichia coli. J. Mol. Biol., 231, 141–144 (1993).
13) Shiio, I., Ozaki, H., and Ujigawa, K., Regulation of
citrate synthase in Brevibacterium flavum, a glutamate-
producing bacterium. J. Biochem., 82, 395–405 (1977).
14) Shiio, I., and Ozaki, H., Concerted inhibition of
isocitrate dehydrogenase by glyoxylate plus oxalacetate.
J. Biochem., 64, 45–53 (1968).
In conclusion, fumarase of C. glutamicum possesses
the same characteristics as pig-heart fumarase and
E. coli FumC in many biochemical respects, but features
no inhibition by citrate and slight inhibition by pyro-
mellitic acid (a three-order larger Ki value). The point of
difference from the pig-heart enzyme is that C. gluta-
micum fumarase was not inhibited by succinate, L-
tartrate, glycine, or maleate (Table 3). Such a difference
in kinetic properties must come from the steric variety of
the active center of the enzyme molecule. With a view to
unraveling the reaction mechanism in the active center,
it was of interest to investigate substrate and substrate-
analog binding with C. glutamicum fumarase by crys-
tallographic, kinetic, and site-directed mutagenesis ex-
periments, referring to recent studies with fumarases of
pig heart,20,40) E. coli,21,41) and S. cerevisiae.22,42)
Concerning the slight activation of C. glutamicum
fumarase by Mg2þ, as described above, we noticed a
suggestion by Weaver and Banaszak21) that a ‘‘Mg2þ
site’’ might be present at the same site of a bound water
molecule (W-26) in the region of the active center of
E. coli fumarase.
15) Ozaki, H., and Shiio, I., Regulation of the TCA and
glyoxylate cycles in Brevibacterium flavum. I. Inhibition
of isocitrate lyase and isocitrate dehydrogenase by
organic acids related to the TCA and glyoxylate cycles.
J. Biochem., 64, 355–363 (1968).
16) Shiio, I., and Ujigawa-Takeda, K., Presence and regu-
lation of ꢀ-ketoglutarate dehydrogenase complex in a
glutamate-producing bacterium, Brevibacterium flavum.
Agric. Biol. Chem., 44, 1897–1904 (1980).
Acknowledgment
17) Genda, T., Nakamatsu, T., and Ozaki, H., Purification
and characterization of malate dehydrogenase from
Corynebacterium glutamicum. J. Biosci. Bioeng., 95,
562–566 (2003).
The authors thank Dr. Yoshimi Yamamoto of the
College of Agriculture of Yamaguchi University for
technical advice in enzyme preparation.
18) Molenaar, D., van der Rest, M. E., and Petrovic, S.,
Biochemical and genetic characterization of the mem-
brane-associated malate dehydrogenase (acceptor) from
Corynebacterium glutamicum. Eur. J. Biochem., 254,
395–403 (1998).
19) Molenaar, D., van der Rest, M. E., Drysch, A., and
Yucel, R., Functions of the membrane-associated and
cytoplasmic malate dehydrogenases in the citric acid
cycle of Corynebacterium glutamicum. J. Bacteriol.,
182, 6884–6891 (2000).
20) Beeckmans, S., and Driessche, E. V., Pig heart fumarase
contains two distinct substrate-binding sites differing in
affinity. J. Biol. Chem., 273, 31661–31669 (1998).
21) Weaver, T., and Banaszak, L., Crystallographic studies
of the catalytic and a second site in fumarase C from
Escherichia coli. Biochemistry, 35, 13955–13965
(1996).
22) Weaver, T., Lees, M., Zaitsev, V., Zaitseva, I., Duke, E.,
Lindley, P., McSweeny, S., Svensson, A., Keruchenko,
J., Keruchenko, I., Gladilin, K., and Banaszak, L.,
Crystal structures of native and recombinant yeast
fumarase. J. Mol. Biol., 280, 431–442 (1998).
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