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 MnCl 2, 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 of 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.