Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.

Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase.

Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.

Structural and enzymatic properties of adenine 1-oxide nucleotides.

We decribed the preparation of adenine 1-oxide nucleotides by oxidation of the natural compounds with monopermaleic acid in aqueous solutions at neutral pH, with an overall yield after chromatographic purification between 75 and 80%. If irradiated, the adenine 1-oxide nucleotides undergo a photochemical rearrangement reaction, the main photoproducts in aqueous solution at alkaline pH being the corresponding isoguanine nucleotides. The modified ring vibration pattern of the 1-oxide analogues as well as the 13C chemical shift indicate a loss of aromaticity as compared to the natural compounds. Coupling constant measurements show that the dihedral angle between the 31POC and OC13C planes is around 180degree, i.e., trans, as in the natural adenine nucleotides. The modified adenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes, as substrates of varous phosphotransferases from mitochondria or cytosol, and as allosteric effectors in the reactions catalyzed by glutamate dehydrogenase and phosphofructokinase. Although the adenine 1-oxide nucleotides are not recognized by the translocase system of the inner mitochondrial membrane, they are good substrates for mitochondrial phosphotransferases located in the intermembrane space. Similarly, they participate in the phosphoryl group transfer reactions catalyzed by pyruvate kinase, phosphofructokinase, and hexokinase. As allosteric effectors, the modified nucleotides are less active than the natural compounds, probably because of a lower binding capacity to the allosteric sites of the regulatory enzymes.

Participation of N1-oxide derivatives of adenine nucleotides in the phosphotransferase activity of liver mitochondria.

The modified adenine nucleotides ATP-NO, ADP-NO, and AMP-NO were tested as potential substrates and/or inhibitors of mitochondrial phosphotransferases. ADP-NO is not recognized by the translocase system located in the inner mitochondrial membrane; however, it is rapidly phosphorylated to ATP-NO in the outer compartment of mitochondria, by way of the nucleosidediphosphate kinase (EC 2.7.4.6) reaction, provided there is sufficient ATP in the mitochondria. AMP-NO is not phosphorylated by liver mitochondria to the corresponding nucleoside diphosphate; it cannot serve as substrate for adenylate kinase (EC 2.7.4.3). ATP-NO and ADP-NO, however, are substrates of this enzyme. The apparent equilibrium constant for the reaction, ADP-NO + ADP right harpoon over left harpoon ATP-NO + AMP, of 0.908 at pH 7.4 and 5 mM Mg(2+) is significantly higher than that of the reaction with natural nucleotides. Although adenosine N(1)-oxide is easily phosphorylated to AMP-NO by adenosine kinase [Schnebli et al. (1967) J. Biol. Chem. 242, 1997-2004], the formation of corresponding nucleoside triphosphate in vivo seems also to be limited by adenylate kinase; adenosine N(1)-oxide cannot replace adenosine in restoring the normal ATP level in ethionine-treated rats.