The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.
Frenkel R (1975) Regulation and physiological functions of malic enzymes. Curr Top Cell Regul 9: 157–181.
[3]
Edens WA, Urbauer JL, Cleland WW (1997) Determination of the chemical mechanism of malic enzyme by isotope effects. Biochemistry 36: 1141–1147.
[4]
Xu Y, Bhargava G, Wu H, Loeber G, Tong L (1999) Crystal structure of human mitochondrial NAD(P)(+)-dependent malic enzyme: a new class of oxidative decarboxylases. Structure 7: 877–889.
[5]
Chang GG, Tong L (2003) Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 42: 12721–12733.
[6]
Sauer LA, Dauchy RT (1978) Identification and properties of the nicotinamide adenine dinucleotide (phosphate)+-dependent malic enzyme in mouse ascites tumor mitochondria. Cancer Res 38: 1751–1756.
[7]
Sauer LA, Dauchy RT, Nagel WO, Morris HP (1980) Mitochondrial malic enzymes. Mitochondrial NAD(P)+-dependent malic enzyme activity and malate-dependent pyruvate formation are progression-linked in Morris hematomas. J Biol Chem 255: 3844–3848.
[8]
Cohen PT, Omenn GS (1972) Human malic enzyme: high-frequency polymorphism of the mitochondrial form. Biochem Genet 7: 303–311.
[9]
Povey S, Wilson DE Jr, Harris H, Gormley IP, Perry P, et al. (1975) Sub-unit structure of soluble and mitochondrial malic enzyme: demonstration of human mitochondrial enzyme in human-mouse hybrids. Ann Hum Genet 39: 203–212.
[10]
Loeber G, Dworkin MB, Infante A, Ahorn H (1994) Characterization of cytosolic malic enzyme in human tumor cells. FEBS Lett 344: 181–186.
[11]
Chang GG, Wang JK, Huang TM, Lee HJ, Chou WY, et al. (1991) Purification and characterization of the cytosolic NADP(+)-dependent malic enzyme from human breast cancer cell line. Eur J Biochem 202: 681–688.
[12]
Loeber G, Maurer-Fogy I, Schwendenwein R (1994) Purification, cDNA cloning and heterologous expression of the human mitochondrial NADP(+)-dependent malic enzyme. Biochem J 304: 687–692.
[13]
Sanz N, Díez-Fernández C, Valverde AM, Lorenzo M, Benito M, et al. (1997) Malic enzyme and glucose 6-phosphate dehydrogenase gene expression increases in rat liver cirrhogenesis. Br J Cancer 75: 487–492.
[14]
Zhang YJ, Wang Z, Sprous D, Nabioullin R (2006) In silico design and synthesis of piperazine-1-pyrrolidine-2,5-dione scaffold-based novel malic enzyme inhibitors. Bioorg Med Chem Lett 16: 525–528.
[15]
Zelewski M, Swierczyński J (1991) Malic enzyme in human liver. Intracellular distribution, purification and properties of cytosolic isozyme. Eur J Biochem 201: 339–345.
[16]
Kam PL, Lin CC, Li JC, Meng CL, Chang GG (1988) High malic enzyme activity in tumor cells and its cross-reaction with anti-pigeon liver malic enzyme serum. Mol Cell Biochem 79: 171–179.
[17]
Wise EM Jr, Ball EG (1964) Malic enzyme and lipogenesis. Proc Natl Acad Sci USA 52: 1255–1263.
[18]
Fitch WM, Chaikoff IL (1960) Extent and patterns of adaptation of enzyme activities in livers of normal rats fed diets high in glucose and fructose. J Biol Chem 235: 554–557.
[19]
Crozier G, Bois-Joyeux B, Chanez M, Girard J, Peret J (1987) Metabolic effects induced by long-term feeding of medium-chain triglycerides in the rat. Metabolism 36: 807–814.
[20]
van Schothorst EM, Keijer J, Pennings JL, Opperhuizen A, van den Brom CE, et al. (2006) Adipose gene expression response of lean and obese mice to short-term dietary restriction. Obesity 14: 974–979.
[21]
Yen TT, Greenberg MM, Yu PL, Pearson DV (1976) An analysis of the relationships among obesity, plasma insulin and hepatic lipogenic enzymes in “viable yellow obese” mice (Avy/a). Horm Metab Res 8: 159–166.
[22]
Huupponen R, Karvonen I, Sotaniemi E (1989) Activity of hepatic glucose phosphorylating and NADPH generating enzymes in Zucker rats. Diabetes Res 10: 143–146.
[23]
Ayala A, F-Lobato M, Machado A (1986) Malic enzyme levels are increased by the activation of NADPH-consuming pathways: detoxification processes. FEBS Lett 202: 102–106.
[24]
Yang Z, Lanks CW, Tong L (2002) Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate. Structure 10: 951–960.
[25]
Yang Z, Zhang H, Hung HC, Kuo CC, Tsai LC, et al. (2002) Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme. Protein Sci 11: 332–341.
[26]
Yang Z, Floyd DL, Loeber G, Tong L (2000) Structure of a closed form of human malic enzyme and implications for catalytic mechanism. Nat Struct Biol 7: 251–257.
[27]
Coleman DE, Rao GS, Goldsmith EJ, Cook PF, Harris BG (2002) Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution. Biochemistry 41: 6928–6938.
[28]
Rao GS, Coleman DE, Karsten WE, Cook PF, Harris BG (2003) Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site. J Biol Chem 278: 38051–38058.
[29]
Tao X, Yang Z, Tong L (2003) Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism. Structure 11: 1141–1150.
[30]
Hsieh JY, Chen SH, Hung HC (2009) Functional roles of the tetramer organization of malic enzyme. J Biol Chem 284: 18096–18105.
[31]
Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripas' AF, et al. (1991) Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31: 119–128.
[32]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[33]
Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131: 266–280.
[34]
Schuck P, Perugini MA, Gonzales NR, Howlett GJ, Schubert D (2002) Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys J 82: 1096–1111.
