Modification of pig liver dimeric dihydrodiol dehydrogenase with diethylpyrocarbonate and by rose bengal-sensitized photooxidation: evidence for an active-site histidine residue.
Dihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.
Shinoda
M
M
Gihoku General Hospital, Gifu.
Hara
A
A
Nakayama
T
T
Deyashiki
Y
Y
Sawada
H
H
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010777:Photochemistry; D012395:Rose Bengal / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014443:Tyrosine
D006898:Hydroxylamines; D046911:Macromolecular Substances; D012395:Rose Bengal; D019811:Hydroxylamine; D014443:Tyrosine; D006639:Histidine; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase; D004047:Diethyl Pyrocarbonate