[123] Nicotinamide deamidase

Su, Shirley; Chaykin, Sterling

doi:10.1016/s0076-6879(71)18080-9

Cited by 6 publications

(4 citation statements)

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“…This assay is slightly modified from that described by Su et al (35). Typical assay mixtures contained 1.25 µM to 3.2 mM nicotinamide or analog, 0.2 mM NADPH, 3.3 mM α-ketoglutarate, 50 nM to 10 µM Pnc1 WT or mutant, and 3 units of glutamate dehydrogenase from bovine liver in 50 mM sodium phosphate at pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

“…Nicotinamidase activity was measured continuously using an enzyme-coupled assay with glutamate dehydrogenase using a Multiskan Ascent microplate reader (LabSystems, Franklin, MA). This assay is slightly modified from that described by Su et al 35 Typical assay mixtures contained 1.25 μM to 3.2 mM nicotinamide or analogue, 0.2 mM NADPH, 3.3 mM α-ketoglutarate, 50 nM to 10 μM wild-type (WT) or mutant Pnc1, and 3 units of glutamate dehydrogenase from bovine liver in 50 mM sodium phosphate (pH 7.5). Assays were conducted in a final volume of 300 μL per well in a clear, flat-bottom, 96-well plate.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

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Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

et al. 2011

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Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD+ salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs acting as inhibitors or substrates were examined revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15N and 13C kinetic isotope effects (KIE) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was observed indicative of a higher commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

et al. 2011

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“…The microsomal enzyme has been purified and characterized (Su et al, 1969;Su and Chaykin, 1971). Two different MI protein fractions endowed with deamidase activity have been isolated.…”

Section: Nicotinamide Deamidasementioning

confidence: 99%

“…Two different MI protein fractions endowed with deamidase activity have been isolated. The fraction of 218,000 M, used for the characterization is a glycoprotein containing 2.84% mannose and 0.57% glucosamine, consisting of four subunits of 60,000 MI (Su and Chaykin, 1971;Gillam et al, 1973). It is inhibited by a large variety of compounds, including fatty acids present in liver homogenates (Greengard et d., 1969;Gillam et al, 1973), thyroxine, which causes the disruption of the enzyme structure (Gillam et al, 1973), and the site-specific reagents DFP (diisopropylfluorophosphate) and ZPCK (carbobenzoxyamido-2-phenylethyl chloromethyl ketone), thus implicating the presence of seryl and histidil residues in the active center (Albizati, and Hedrick, 1972;Gillam et al, 1973).…”

Section: Nicotinamide Deamidasementioning

confidence: 99%

Enzymology of Nad ⁺ Synthesis

Magni

Amici

Emanuelli

et al. 1999

Advances in Enzymology - And Related Areas of Molecular Biology

195

194

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The pyridine-nucleotide cycle in tobacco

Wagner¹,

Feth²,

Wagner³

1986

Planta

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In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NAD→NMN→nicotinamide→nicotinic acid→NaMN→nicotinic acid adenine dinucleotide→NAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.

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[123] Nicotinamide deamidase

Cited by 6 publications

References 5 publications

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Enzymology of Nad ⁺ Synthesis

The pyridine-nucleotide cycle in tobacco

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[123] Nicotinamide deamidase

Cited by 6 publications

References 5 publications

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Enzymology of Nad + Synthesis

The pyridine-nucleotide cycle in tobacco

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Enzymology of Nad ⁺ Synthesis