Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

Abstract

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8. and the active enzyme was expressed in Eschericia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from β-galactosidase coded by the lac Z' gene C99S and R70Q mutants of the enzyme were generated by the M 13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation L-malate at pH 7.0: K(m) NADP+ (4.6 μM); K(m) L-malate (73 μM); K(cat) t (160 s -1); K(a) (2.4/μM) and Ka' (270 μM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of k(cat) at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged K(m) for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, K(a)'); however. k(cat) decreased 3-fold and K(m) for L-malate and K(a) each increased 4-fold, resulting in a catalytic efficiency [k(cat)/(K(m) NADP+ x K(m) L-malate x K(a))] equal to 3.7% of the natural duck enzyme. These resu]ts suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.