title: Duck liver malic enzyme: sequence of a tryptic peptide containing the cysteine residue labeled by the substrate analog bromopyruvate.
abstract: Malic enzyme of duck liver is alkylated by bromopyruvate with half-of-the-sites stoichiometry, and with accompanying loss of oxidative decarboxylase and enhancement of pyruvate reductase activities as was previously shown for the pigeon enzyme (Hsu, R.Y. (1982) Mol. Cell. Biochem. 43, 3-26). In the present work, the alkylated enzyme is shown to bind NADPH, but not L-malate in the presence of MnCl2, indicating impairment of the enzyme site for the substrate and/or divalent metal. The enzyme was differentially labeled by 3-bromo-1-[14C]-pyruvate and digested with TPCK-treated trypsin. Two peptides bearing the susceptible residue were purified by high-performance liquid chromatography and sequenced. Peptide II has the sequence of FMPIVYTPTVGLAXQQYGLAFR, corresponding to residues 86-107 (temporary numbering) of the duck enzyme; cysteine-99(x) is not detected, indicating that it is the target of modification by bromopyruvate. Peptide I is a truncated form of peptide II lacking five amino acid residues at the C-terminal. Cysteine-99 is conserved in malic enzymes from duck, rat, mouse, maize, human, Flaveria trinervia and Bacillus stearothermophilus.
authors.affiliation: Health Science Center, State University of New York, Syracuse.
authors.forename: J
authors.initials: J
authors.lastname: Satterlee
authors.forename: R Y
authors.initials: RY
authors.lastname: Hsu
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D004372:Ducks; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D010446:Peptide Fragments; D011773:Pyruvates; C017092:bromopyruvate; D008291:Malate Dehydrogenase; D014357:Trypsin