Inactivation of rat ovarian 20 alpha-hydroxysteroid dehydrogenase by N-alkylmaleimides.
A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20 alpha-hydroxysteroid dehydrogenase at pH 7.7, 25 degrees C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the Kd values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min-1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20 alpha-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pKa near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.
D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005452:Fluoresceins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D009941:Organomercury Compounds / Q000494:pharmacology; D010053:Ovary / Q000201:enzymology*; D010422:Pentanones / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D005452:Fluoresceins; D008301:Maleimides; D009941:Organomercury Compounds; D010422:Pentanones; C001673:fluorescein mercuric acetate; C008790:acetylacetone; C039995:3-aminopyridine-1,N(6)-ethenoadenine dinucleotide phosphate; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D005033:Ethylmaleimide