nicotinate-mononucleotide and phthalic-acid

Quinolinic acid phosphoribosyltransferase (NadC; EC 2.4.2.19) is the key enzyme of NAD(+) biosynthesis in both prokaryotes and eukaryotes. NadC catalyzes the decarboxylation of quinolinic acid (QA) to produce nicotinic acid mononucleotide (NAMN), an intermediate in NAD synthesis. NadCs of Helicobacter pylori appeared to be a hexamer during the purification procedure. Three different complexes of NadC, with QA, NAMN and phthalic acid (PA), an analogue of QA, were crystallized at 294 +/- 1 K using the hanging-drop vapour-diffusion method. The QA complex crystal was found to belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 148.8, c = 145.7 A, alpha = beta = gamma = 90 degrees. Diffraction data were collected from the NadC-substrate and NadC-substrate analogue complexes to resolutions of 2.3 A (QA), 2.8 A (PA) and 3.3 A (NAMN) using synchrotron X-ray radiation.

Topics: Cloning, Molecular; Crystallization; Crystallography, X-Ray; Helicobacter pylori; Nicotinamide Mononucleotide; Pentosyltransferases; Phthalic Acids; Protein Binding; Quinolinic Acid

Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 +/- 0.30 microM for quinolinic acid and Km = 65.13 +/- 13.74 microM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 +/- 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 +/- 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a greater than 20-fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippo-campus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve-ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at -80 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Brain; Kinetics; Liver; Nicotinamide Mononucleotide; Pentosyltransferases; Phosphoribosyl Pyrophosphate; Phthalic Acids; Quinolinic Acid; Quinolinic Acids; Rats; Rats, Inbred Strains; Retina; Substrate Specificity; Tissue Distribution