Biosynthesis of Diphosphopyridine Nucleotide

The enzyme diphosphopyridine nucleotide synthetase (deamido-DPN: ammonia amido-ligase (AMP)), was purified about 450-fold from a crude extract from Escherichia coli by protamine sulfate fractionation, ammonium sulfate precipitation, and diethylaminoethyl cellulose chromatography. The reaction catalyzed by DPN synthetase in E. coli was shown to be ATP + deamido-DPN + NH3→DPN + AMP + PPi The enzyme is specific for ATP. Mg++ or Mn++ could satisfy the metal ion requirement. Ammonia was shown to be far superior to l-glutamine as the nitrogen donor for the enzyme. The apparent Km for ammonia was 6.5 x 10-5 m while that for l-glutamine was 1.6 x 10-2 m. Azaserine, an inhibitor of the DPN synthetase (deamido-DPN:glutamine amido-ligase (AMP)) from yeast, did not inhibit the E. coli DPN synthetase. On the other hand, psicofuranine (9-d-psicofuranosyl-6-aminopurine), which was an effective inhibitor of the E. coli DPN synthetase did not inhibit the yeast DPN synthetase. The kinetics and properties of the psicofuranine inhibition were studied.