DUCK LIVER MALIC ENZYME - EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE WILD-TYPE ENZYME AND SITE-DIRECTED MUTANTS

A cDNA for duck liver 'malic' enzyme (EC 1. 1. 1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M 1 3 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 of L-malate at pH 7.0: K(m) NADP+ (4.6-mu-M); K(m) L-malate (73-mu-M); k(cat) (160 s-1); K(a) (2.4-mu-M) and K(a)' (270-mu-M), 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 Km L-malate x K(a))] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.