Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

Abstract

A cDNA coding for human breast cancer cell cytosolic NADP⁺-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3′-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994),FEBS Lett. 344, 181–186] reveals that three nucleotides in the open reading frame and the length of 3′-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally inEscherichia coli cells. ItsK _m value forl-malate (1.21±0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29±0.04 mM) but similar to that for the full-length recombinant enzyme (1.06±0.07 mM). TheK _m values for Mn²⁺ and NADP⁺ (0.26±0.03 and 0.97±0.4μM, respectively) are similar to those for the natural enzyme (0.12±0.02 and 1.9±0.3μM, respectively) or the recombinant wild-type enzyme (0.56±0.04 and 0.44±0.02μM, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. TheK _m values forl-malate and Mn²⁺ of the truncated enzyme (11.2±0.9 mM and 61.2±4.6μM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21±0.02 mM and 1.06±0.08μM, respectively) or the recombinant wild-type enzyme (0.25±0.01 mM and 1.48±0.05μM, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.