Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes.
Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.
D050788:Alanine Dehydrogenase; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013552:Swine
D009243:NAD; D010088:Oxidoreductases; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D005969:Glutamate Dehydrogenase