Long-range interaction between the enzyme active site and a distant allosteric site in the human mitochondrial NAD(P)+-dependent malic enzyme.
Our previous study has suggested that mutation of the amino acid residue Asp102 has a significant effect on the fumarate-mediated activation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME). In this paper, we examine the cationic amino acid residue Arg98, which is adjacent to Asp102 and is highly conserved in most m-NAD(P)-MEs. A series of R98/D102 mutants were created to examine the possible interactions between Arg98 and Asp102 using the double-mutant cycle analysis. Kinetic analysis revealed that the catalytic efficiency of the enzyme was severely affected by mutating both Arg98 and Asp102 residues. However, the binding energy of these mutant enzymes to fumarate as determined by analysis of the K(A,Fum) values, show insignificant differences, indicating that the mutation of Arg98 and Asp102 did not cause a significant decrease in the binding affinity of fumarate. The overall coupling energies for R98K/D102N as determined by analysis of the k(cat)/K(m) and K(A,Fum) values were -2.95 and -0.32kcal/mol, respectively. According to these results, we conclude that substitution of both Arg98 and Asp102 residues has a synergistic effect on the catalytic ability of the enzyme.
Hsieh
Ju-Yi
JY
Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan.
Su
Kuo-Liang
KL
Ho
Pei-Tzu
PT
Hung
Hui-Chih
HC
D000495:Allosteric Site / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005650:Fumarates / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013816:Thermodynamics
D017931:DNA Primers; D005650:Fumarates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001120:Arginine; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)