Nicotinamide 3,N⁴-ethenocytosine dinucleotide, an analog of nicotinamide adenine dinucleotide. Synthesis and enzyme studies

Abstract

A structural analog of NAD+, NICOTINAMIDE 3,N-4ethenocytosine dinucleotide (epsilonNCD+), has been synthesized, characterized, and compared in activity with the natural coenzyme in several enzyme systems. The Vmax and apparent Km values were determined for NAD+, epsilonNCD+, and epsilonNAD+ (nicotinamide 1, N6-ethenoadenine dinucleotide) with yeast alcohol, horse liver alcohol, pig heart malate, beef liver glutamate, and rabbit muscle lactate and glyceraldehyde-3-phosphate dehydrogenases. The Vmax for epsilonNCD+ was as great or greater than that obtained for NAD+ with three of the enzymes, 60-80 per cent with two others, and 14 percent with one. EpsilonNCD+ was found to be more active than epsilonNAD+ with all six dehydrogenases. EpsilonNCD+ served as a substrate for Neurospora crassa tnadase, but could not be phosphorylated with pigeon liver NAD+ kinase. NAD+ pyrophosphorylase from pig liver was unable to catalyze the formation of epsilonNCD+ from the triphosphate derivative of epsilon-cytidine and nicotinamide mononucleotide, but was able to slowly catalyze the pyrolytic cleavage of epsilonNCD+. The coenzyme activity of epsilonNCD+ with dehydrogenases can be discussed in terms of the close spatial homology of epsilonNCD+ and NAD+, which may allow similar accommodations within the enzyme binding regions.

Dynamic and static quenching of 1,N6-ethenoadenine fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3-(indol-3-yl) propyl) adenine.

“…While the fluorescence lifetime of ENAD+ is 9.2% of the value for EAMP, the relative quantum yield is only 5% of that observed after snake venom treatment, taking into account a correction for hypochromism in the dinucleotide, an observation which is also consistent with a folded structure (22,(37)(38)(39)(40)(41)(42).…”

Reactions at the termini of tRNA with T4 RNA ligase

“…Descending paper chromatography was carried out on Whatman 3 MM paper using either a 1:1 (solvent A) or a 7:3 (solvent B) V/V mixture of 1 M ammonium acetate pH 7.0 and 95% ethanol. Base hydrolysis of P labeled tRNA was carried out in 0.3 N KOH at 37°C for 16 hours, the mononucleotides were adsorbed to charcoal and removed with 50% ethanol containing 0.15 M NH40H (20). The mononucleotides were separated on Whatman #1 paper by flat plate high voltage electrophoresis (Savant FP-30A) at 65 volts/cm for 2 hour in 25 mM sodium citrate pH 4.0.…”

Trimethylene bridges as synthetic spacers for the detection of intramolecular interactions

“…Details of the dehydrogenase activities observed using the modified coenzymes eNAD+ and eNCD+, which are of interest in connection with other applications of structural and fluorescent modification, are to be found elsewhere. 74,75 Finally, model compounds, Biot-C3-Ind and Biot-C4-Ind, having triand tetramethylene bridges between the biotin ring system and indole were synthesized to help uncover the basis of the strong binding of biotin to avidin, one of the tightest biological complexes known (KD = ca. 10"15 M), and to identify any contributory biotin-tryptophan interaction.…”