Saccharopine Dehydrogenase: A KINETIC STUDY OF COENZYME BINDING

Abstract

In addition to a high degree of specificity toward its amino acid and keto acid substrates, saccharopine dehydrogenase (e-N-( l -glutaryl-2)- l -lysine: NAD oxidoreductase (lysine-forming)) showed a strict coenzyme specificity. NADP+ did not support the reaction in the direction of oxidative cleavage of saccharopine, and NADPH was a poor coenzyme in the reverse direction. NADPH was bound by the enzyme with much less affinity than NADH, and its binding resulted in a large increase in Michaelis constants for substrates which add subsequently. Examination with the compounds that are fragments of the coenzyme (NAD+ or NADH) molecule revealed that adenosine was the smallest fragment that inhibited the reaction. The presence of a phosphate or pyrophosphate group at the 5′ position of the adenosine moiety greatly increased, while the presence of phosphate at or deletion of the oxygen from the 2′ position decreased the inhibitory action. NMN+, NMNH, guanine and hypoxanthine nucleotides, and pyrimidine nucleotides were not inhibitory. Kinetic analyses of the inhibition by AMP, ADP, ADP ribose, and ATP showed that they were competitive with respect to the coenzyme, and noncompetitive with respect to the other substrates, and indicate that they are bound at the coenzyme-binding site. The apparent inhibition constants were not altered by the variation in the concentrations of the second substrates, suggesting that the binding of these nucleotides was not followed by the combination of substrates.