Purification and Properties of Yeast Nicotinamide Adenine Dinucleotide Synthetase

• Published in: The Journal of biological chemistry Vol. 247; no. 15; pp. 4794 - 4802
• Main Authors: Yi, C K, Dietrich, L S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.08.1972
• Subjects: Adenosine Diphosphate - analysis; Adenosine Monophosphate - analysis; Adenosine Triphosphate - analysis; Amino Acids - analysis; Carbon Isotopes; Chemical Phenomena; Chemistry; Chloromercuribenzoates; Chromatography, Ion Exchange; Chromatography, Paper; Cobalt; Drug Stability; Electrophoresis, Disc; Glutamine; Isoflurophate; Kinetics; Ligases - analysis; Ligases - antagonists & inhibitors; Ligases - isolation & purification; Magnesium; Manganese; Molecular Weight; NAD; Phosphorus Isotopes; Time Factors; Yeasts - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)44981-8

Yeast NAD synthetase was purified 2,000-fold. The purified enzyme gave one protein band on disc gel electrophoresis and was found to be monodisperse on high speed equilibrium ultracentrifugation with an apparent molecular weight for the "native" enzyme of 630,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of apparent nonidentical subunits with molecular weights of 80,000 and 65,000. The amino acid composition of "native" enzyme revealed no unusual amino acid residues. The specificity of the substrates, nucleoside triphosphate requirement, and divalent and monovalent metal ion requirements were studied. Nicotinate adenine dinucleotide, but not nicotinate mononucleotide, is the amide acceptor, and glutamine or free ammonia is the amide donor. The enzyme is specific for ATP, exhibiting a stoichiometric cleavage into AMP and PP i during the amidation. Mg 2+ and K + are required for enzymatic activity. Mn 2+ or Co 2+ can replace Mg 2+ to a certain extent. NH 4 + is as effective as K + in stimulating enzymatic activity. The enzyme displays a broad peak of activity between pH 6.2 and 8.4, with maximal activity being observed around pH 7.6 when l -glutamine is used as the amide donor. However, when ammonium chloride is employed as the substrate, the enzymatic activity exhibits a rather narrow peak between pH 8.4 and 8.8. The apparent K m values for nicotinate adenine dinucleotide, ATP, and l -glutamine at pH 7.6 were found to be 1.9 x 10 -4 , 1.7 x 10 -4 , and 5 x 10 -3 m , respectively. The K m for NH 4 Cl at pH 8.6 is 6.4 x 10 -3 m . The catalytic constant was calculated to be approximately 1,260 moles of NAD produced per min per mole of enzyme, based on a molecular weight of 630,000. When the enzyme was previously incubated with azaserine, nicotinate adenine dinucleotide, and ATP, glutamine-dependent activity was progressively inhibited as a function of prior incubation time, whereas with NH 4 Cl as the amide donor there was an initial 20% inhibition and then NH 4 Cl-dependent activity remained constant. p -Chloromercuribenzoate and heavy metals inhibit both glutamine- and NH 4 Cl-dependent activity. Hydroxamate analogues were formed when (nicotinate adenine dinucleotide + ATP) and/or glutamine were incubated with enzyme and hydroxylamine. Hydroxamate analogues were not formed from nicotinate adenine dinucleotide unless ATP was also present. Hydroxamate analogue formation was additive when glutamine, nicotinate adenine dinucleotide, and ATP were added.