Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme.
Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.
Hung
Hui-Chih
HC
Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan. hchung@dragon.nchu.edu.tw
Chien
Yu-Ching
YC
Hsieh
Ju-Yi
JY
Chang
Gu-Gang
GG
Liu
Guang-Yaw
GY
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D005650:Fumarates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D005650:Fumarates; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D009243:NAD; C032005:fumaric acid; D000255:Adenosine Triphosphate; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)