Self-inactivation of an erythrocyte NAD glycohydrolase

Summary NAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N⁶ ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative S for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.This work was supported by Research Grant PCM 76-05839 from the National Science Foundation.