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 M13 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: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. 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 Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] 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.
Hsu
R Y
RY
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Glynias
M J
MJ
Satterlee
J
J
Feeney
R
R
Clarke
A R
AR
Emery
D C
DC
Roe
B A
BA
Wilson
R K
RK
Goodridge
A G
AG
Holbrook
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004372:Ducks; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D008291:Malate Dehydrogenase