Tohoku J. Exp. Med., 1997, 181 (4)

4 Pro-R Hydrogen of NADPH was Abstracted for Enzymatic Hydride Transfer by N-Ethylmaleimide Reductase of Yarrowia lipolytica

MICHINAO MIZUGAKI, KOICHI MIURA, HIROTAKA YAMAMOTO, MICHIKO KAYABA-NAKAZAWA, KIYOTO EDO, YOSHIHISA TOMIOKA and TAKANORI HISHINUMA

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-77

  • We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y.lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y.lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y.lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y.lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.
    Key words--- N-ethylmaleimide metabolism; oxidoreductase antagonist and inhibitor; unsaturated fatty acids

    Tohoku J. Exp. Med., 1997, 181, 447-457
    Address for reprints: Michinao Mizugaki, Ph.D., Professor and Director, Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryomachi, Aoba-ku, Sendai 980-77, Japan.

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