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J. Biochem, 1992, Vol. 112, No. 6 834-839
© 1992 Japanese Biochemical Society

research-article

Modification of Pig Liver Dimeric Dihydrodiol Dehydrogenase with Diethylpyrocarbonate and by Rose Bengal-Sensitized Photooxidation: Evidence for an Active-Site Histidine Residue1

Michio Shinoda*, Akira Hara**,2, Toshihiro Nakayama**, Yoshihiro Deyashiki** and Hideo Sawada***

*Gihoku General Hospital Takatomi, Gifu 501–21
**Laboratory of Biochemistry, Department of Manufacturing Pharmacy, Gifu Pharmaceutical University Mitahora-higashi, Gifu, Gifu 502
***Gifu College of Medical Technology Seki, Gifu

2 To whom correspondence should be addressed

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.

1This study was supported in part by a grant for Cooperative Research from the Primate Research Institute, Kyoto University.


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