Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell.
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 in Escherichia coli cells. Its Km value for L-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). The Km values for Mn2+ and NADP+ (0.26 +/- 0.03 and 0.97 +/- 0.4 microM, respectively) are similar to those for the natural enzyme (0.12 +/- 0.02 and 1.9 +/- 0.3 microM, respectively) or the recombinant wild-type enzyme (0.56 +/- 0.04 and 0.44 +/- 0.02 microM, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. The Km values for L-malate and Mn2+ of the truncated enzyme (11.2 +/- 0.9 mM and 61.2 +/- 4.6 microM, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21 +/- 0.02 mM and 1.06 +/- 0.08 microM, respectively) or the recombinant wild-type enzyme (0.25 +/- 0.01 mM and 1.48 +/- 0.05 microM, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.
Chou
W Y
WY
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Huang
S M
SM
Chang
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D003001:Cloning, Molecular; D010856:Columbidae; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D015342:DNA Probes; D018076:DNA, Complementary / Q000737:chemistry; D004273:DNA, Neoplasm / Q000737:chemistry; D005260:Female; D006801:Humans; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)