title: The role of cysteine in the alteration of bovine liver dihydrodiol dehydrogenase 3 activity.
abstract: Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extremely 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, NAD+, nicotinamide mononucleotide or 5'-ADP.DD3 was absorbed by an affinity column of thiopropyl-Sepharose 6B, but enzyme incubated with both NEM and NADP+ was not. Moreover, one [14C]NEM molecule was incorporated into a cysteine of DD3 in the presence, and two cysteines of DD3 in the absence, of NADP+. These results suggested that two cysteine residues were modified per enzyme molecule by NEM, one was protected by NADP+ and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the digestion with lysyl endopeptidase of [14C]NEM-modified DD3 could be separated by reverse-phase HPLC. P1, which was radiolabelled by [14C]NEM only 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 was very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also highly 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 study 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 through 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 cysteine residue corresponding to Cys-145 of rat 3 alpha-HSD/DD, may be located near the surface of the enzyme molecule.
authors.affiliation: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nanjo
authors.forename: H
authors.initials: H
authors.lastname: Adachi
authors.forename: M
authors.initials: M
authors.lastname: Aketa
authors.forename: T
authors.initials: T
authors.lastname: Mizoguchi
authors.forename: T
authors.initials: T
authors.lastname: Nishihara
authors.forename: T
authors.initials: T
authors.lastname: Terada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005033:Ethylmaleimide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D007527:Isoenzymes; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase; D003545:Cysteine; D005033:Ethylmaleimide