Involvement of Phe19 in the Mn(2+)-L-malate binding and the subunit interactions of pigeon liver malic enzyme.
A triple mutant, F19S/N250S/L353Q, of pigeon liver malic enzyme was found to have no detectable enzymatic activity [Chou, W.-Y., Huang, S.-M., & Chang, G.-G. (1994) Arch. Biochem. Biophys. 310, 158-166]. In the present study, point mutants at these positions (F19S, N250S, and L353Q) were prepared by site-directed mutagenesis. Both N250S and L353Q have kinetic properties similar to those of the wild-type. On the other hand, the K(m)(app) values for both Mn2+ and L-malate of F19S were increased by approximately 10-fold, while the kcat value was decreased by 5-fold, which results in a decrease of the apparent catalytic efficiency (kcat/K(mNADP)K(mMal)K(mMn) by approximately 300-fold. These results clearly indicate that the F19S mutation is mainly responsible for the undetectable enzyme activity of the triple mutant. Three more Phe19 mutants (F19Y, F19G, and F19A) were then prepared. There is a direct correlation between the size of the substitutes and the affinities for Mn2+ and L-malate. The kinetic parameters for F19Y were similar to those for wild-type. Both F19A and F19G reveal a 5-fold decrease of kcat values. Two K(dMn) values for the high- and low-affinity sites, respectively, were detectable for the wild-type. On the contrary, only one K(dMn) value was detected for the F19 mutants, which was increased in the order of F19G > F19A > F19S > F19Y, with F19G being the most affected mutant. The K(mMal) values of F19G and F19A were increased 100- and 6-fold, respectively. The catalytic efficiency (kcat/K(mNADP)K(dMal)K(dMn)) of F19G was decreased to only 0.01% of that of the wild-type. The above results clearly indicate that the hydrophobic aromatic ring at position 19 plays a critical role in L-malate and Mn2+ binding. Furthermore, all mutants that have a small residue at position 19 exist as monomers. Therefore, Phe19 may locate in or near the regions for Mn(2+)-L-malate binding as well as for the subunit contact. These results are compatible with the asymmetric model for the quaternary structure of malic enzyme we proposed previously [Chang, G.-G., Huang, T.-M., Huang, S.-M., & Chou, W.-Y. (1994) Eur. J. Biochem. 225, 1021-1027]. The possible roles of the N-terminus of malic enzyme were also addressed.
Chou
W Y
WY
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Liu
M Y
MY
Huang
S M
SM
Chang
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D010856:Columbidae; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D008293:Malates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008345:Manganese; D010649:Phenylalanine; C030298:malic acid; D008291:Malate Dehydrogenase; C034544:D-malate dehydrogenase (decarboxylating)