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J. Biochem, 1974, Vol. 76, No. 6 1259-1268
© 1974 Japanese Biochemical Society

research-article

Studies on the Regulatory Functions of Malic Enzymes

III. Regulatory Effects of L-Aspartate, Coenzyme A, and Divalent Metal Ions on NAD-linked Malic Enzyme from Escherichia coli*

Mutsuo YAMAGUCHI**, Masanobu TOKUSHIGE, Kanji TAKEO*** and Hirohiko KATSUKI****

Department of Chemistry, Faculty of Science, Kyoto University Kyoto 606

****To whom all correspondence should be addressed

The effects of L-aspartate, CoA, and divalent metal ions on the catalytic and regulatory properties of NAD-linked malic enzyme [EC 1.1.1.38 [EC] ] from Eschenchza coli were studied.

  1. The Km value for L-malate decreased progressively as L-aspartate concentration was increased. The activation constant for L-aspartate also decreased progressively as L-malate concentration was increased. The lowering effect of L-aspartate on the Km value for L-malate was marked at high pH values, whereas the Vmax value was not affected in the pH region tested.
  2. Besides L-aspartate, amino acids such as {alpha}-methyl-DL-aspartate,ß-methyl-DL aspartate, and D-aspartate activated the enzyme. L-Aspartate andß-methyl-DL aspartate exerted inhibitory effects on the enzyme activity at higher concentrations, whereas {alpha}-methyl-DL-aspartate and D-aspartate were not inhibitory. D-Malate, DL methylmalate, DL-tartrate, succinate, L-{alpha}-aminobutyrate, L-glutamate, and L-aspar agine had no effect.
  3. The saturation profile of divalent metal ions exhibited a marked sigmoidicity. L-Aspartate decreased the half-saturation concentration for divalent metal ions, but it did not significantly alter the sigmoidicity of the saturation curve.
  4. CoA acted antagonistically with respect to L-aspartate and strengthened the cooperativity of L-malate.
  5. Divalent metal ions exhibited a protective effect against heat inactivation of the enzyme. L-Aspartate exhibited the highest degree of protection in the presence of divalent metal ions, and CoA was effective to a similar extent. L-Malate was ineffective even in the presence of divalent metal ions.
  6. Upon aging, the enzyme gradually became desensitized to activation by L-aspartate. The sensitivity was, however, restored by the addition of thiol compounds such as dithiothreitol or 2-mercaptoethanol.

These results strongly suggest that L-aspartate and CoA bind with the enzyme at sites distinct from the active site and have regulatory roles.

*This investigation was suported in part by research grants from the Ministry of Education of Japan and the Takeda Science Foundation.

**Present address : Department of Biochemistry, Asahikawa Medial College, Asahikawa.

***Present address: Chest Disease Research Institute Kyoto University, Kyoto.


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