Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides.
A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
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Beier
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Anderson
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Anderson
D000818:Animals; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors*; D008099:Liver / Q000201:enzymology*; D008301:Maleimides / Q000494:pharmacology*; D012756:Sheep; D013329:Structure-Activity Relationship; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D008301:Maleimides; D013438:Sulfhydryl Compounds; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase