Identification of essential amino acids in the active center of thyroidal NAD+ glycohydrolase.

1. Purified thyroidal NAD+ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action. 2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD+ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine. 3. NAD+ and ADP-ribose partially protect against inactivation with similar efficiencies. 4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD+ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione. 5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains. 6. The mechanistic implications of these results are discussed.