Chemical modification of cysteinyl, lysyl and histidyl residues of mouse liver 17 beta-hydroxysteroid dehydrogenase.
Monomeric 17 beta-hydroxysteroid dehydrogenase from mouse liver was rapidly inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) and 2,4,6-trinitrobenzene-1-sulfonate, and the absorption spectra of the inactivated enzymes indicated that cysteine and lysine residues were modified. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of two cysteine residues or one lysine residue per active site. The inactivation by the two reagents was protected by NADP+ and some coenzyme analogs, but not by a steroid substrate, testosterone. Moreover, chemical modification by diethyl pyrocarbonate also produced inactivation of the enzyme, and showed a difference spectrum with a peak at 242 nm characteristic of N-carbethoxyhistidine residues, which decreased with the addition of hydroxylamine. The inactivation by this reagent, following pseudo-first-order kinetics, was protected partially by either NADP+ or testosterone and completely in the presence of both the coenzyme and substrate. The results suggest the presence of essential cysteine and lysine residues at or near the coenzyme-binding site and that of essential histidine residue(s) in the catalytic region of the active site of mouse liver 17 beta-hydroxysteroid dehydrogenase.
Nakayama
T
T
Department of Biochemistry, Gifu Pharmaceutical University, Japan.
Tanabe
H
H
Deyashiki
Y
Y
Shinoda
M
M
Hara
A
A
Sawada
H
H
D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000737:chemistry; D051379:Mice; D011485:Protein Binding; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry
D003067:Coenzymes; D008024:Ligands; D006639:Histidine; D014302:Trinitrobenzenesulfonic Acid; D004228:Dithionitrobenzoic Acid; D015067:17-Hydroxysteroid Dehydrogenases; D008239:Lysine; D003545:Cysteine; D004047:Diethyl Pyrocarbonate