THE ROLE OF CYSTEINE IN THE ALTERATION OF BOVINE LIVER DIHYDRODIOL DEHYDROGENASE-3 ACTIVITY

Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extr emely sensitive to SH reagents such as N-ethylmaleimide (NEM) and 5,5' -dithiobis(2-nitrobenzoic acid). NEM produced time- and concentration- dependent inactivation of DD3 in a pseudo-first-order reaction manner. This inactivation was prevented by NADP(+), 3-acetylpyridine-adenine dinucleotide phosphate, 2',5'-ADP and 2'-AMP but not by substrates, NA D(+), nicotinamide mononucleotide or 5'-ADP. DD3 was adsorbed by an af finity column of thiopropyl-Sepharose 6B, but enzyme incubated with bo th NEM and NADP(+) was not. Moreover, one [C-14]NEM molecule was incor porated into a cysteine-of DD3 in the presence, and two cysteines of D D3 in the absence, of NADP(+). These results suggested that two cystei ne residues were modified per enzyme molecule by NEM, one was protecte d by NADP(+) and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the diges tion with lysyl endopeptidase of [C-14]NEM-modified DD3 could be separ ated by reverse-phase HPLC. P1, which was radiolabelled by [C-14]NEM o nly in the absence of NADP(+), showed the following sequence; H2N-Tyr- Lys-Pro-Val-Xaa-Asn-Gln-Val-Glu-NEM . Cys-His-Pro-Tyr-Phe-Asn-Gln-Ser- Lys-COOH (Xaa indicates a possible cysteine residue). This sequence wa s very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also hig hly conserved in the aldo-keto reductase superfamily. The sequence of P2, which had radioactivity in both the absence and presence of NADP(), also contained an NEM-modified cysteine and was similar in sequence to the regions located in loop A of rat 3 alpha-HSD/DD. The present s tudy suggests that P1, which may have a cysteine residue corresponding to Cys-193 of rat 3 alpha-HSD/DD, functions in the alteration of DD3 activity depending on the modulation of NADP(+)-binding ability throug h a thiol/disulphide exchange reaction similar to that of rat 3 alpha- HSD/DD shown in our previous results; while P2, which may have a cyste ine residue corresponding to Cys-145 of rat 3 alpha-HSD/DD, may be loc ated near the surface of the enzyme molecule.