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Malic enzyme (ME) is a family of enzymes that catalyze a reversible oxidative decarboxylation of L-malate to pyruvate with simultaneous reduction of NAD(P)+ to NAD(P)H. According to the cofactor specificity, the mammalian enzyme can be categorized into three isoforms. The cytosolic (c) and mitochondrial (m) NADP+-dependent MEs utilize NADP+ as the cofactor. The mitochondrial NAD(P)+-dependent ME can use either NAD+ or NADP+ as the cofactor. In addition, the m-NAD(P)-ME isoform can be inhibited by ATP and allosterically activated by fumarate. In this study, we delineated the determinants for cofactor specificity and isoform-specific inhibition among the ME isoforms. Our data strongly suggest that residue 362 is the decisive factor determining cofactor preference. All the mutants containing Q362K (Q362K, K346S1Q362K, Y347K/Q362K, and K346S/Y347K/Q362K) have a larger kcat,NADP value compared with the kcat,NAD value, indicating that the enzyme has changed to use NADP+ as the preferred cofactor. Furthermore, we suggest that Lys-346 in m-NAD(P)-ME is crucial for the isoform-specific ATP inhibition. The enzymes containing the K346S mutation (K346S, K346S1Y347K, K346S/Q362K, and K346S/Y347K/Q362K) are much less inhibited by ATP and have a larger Ki,ATP value. Kinetic analysis also suggests that residue 347 functions in cofactor specificity. Here we demonstrate that the human K346S/Y347K/Q362K m-NAD(P)-ME has completely shifted its cofactor preference to become an NADP+-specific ME. In the triple mutant, Lys-362, Lys-347, and Ser-346 work together and function synergistically to increase the binding affinity for NADP+. [ABSTRACT FROM AUTHOR]

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ~0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained. [ABSTRACT FROM AUTHOR]

Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface, In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P)[sup+]-dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimertetramer equilibrium. The cytosolic NADP[sup+]-dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer. In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the kcat values of mutants decreased with increasing Kd,24 values, indicating that the dissociation of subunits at the dimer or tetramer interfaces significantly affects the enzyme activity. The above results suggest that the tetramer is required for a fully functional m-NAD(P)-ME. Taken together, the analytical ultracentrifugation data and the kinetic analysis of these interface mutants demonstrate the differential role of tetramer organization for the c-NADP-ME and m-NAD(P)-ME isoforms. The regulatory mechanism of m-NAD(P)-ME is closely related to the tetramer formation of this isoform. [ABSTRACT FROM AUTHOR]

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821–825). In this study, we investigate the important role of Gin362 in the transformation of cofactor specificity from NAD+to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+to NADP+ The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4–6-fold and increased by 5–10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gin362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The AG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gin with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gin362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gin362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gin362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gin362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific. [ABSTRACT FROM AUTHOR]