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.
Verhaeghe
M
M
RUCA-Laboratory for Human Biochemistry, University of Antwerp, Belgium.
De Wolf
M
M
Lagrou
A
A
Van Dessel
G
G
Hilderson
H
H
Dierick
W
W
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004396:Coloring Agents; D006639:Histidine / Q000378:metabolism; D007202:Indicators and Reagents; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors; D009244:NAD+ Nucleosidase; D012685:Sepharose / Q000031:analogs & derivatives; D013961:Thyroid Gland / Q000201:enzymology*
D000596:Amino Acids; D004396:Coloring Agents; D007202:Indicators and Reagents; C017842:cibacron blue-sepharose; D006639:Histidine; D012685:Sepharose; D009699:N-Glycosyl Hydrolases; D009244:NAD+ Nucleosidase