Chemical modification of Corynebacterium sarcosine oxidase: role of sulfhydryl and histidyl groups.
Sarcosine oxidase [sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1] from Corynebacterium contained 8 sulfhydryl groups per mol of enzyme as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 0.2% SDS and by titration with p-chloromercuribenzoate (PMB). Among them, 2 groups were easily modified by iodoacetamide (IAA) and the modification resulted in complete loss of enzymatic activity. The inactivation by IAA followed first-order kinetics with respect to IAA concentration. The presence of acetate, a competitive inhibitor (I), protected the enzyme from inactivation by IAA. However, the protection was only approximately 50%. The enzyme was also inactivated by PMB, but in this case, there was practically no recovery of activity after treatment with thiol compounds. The enzyme was also rapidly inactivated by incubation with diethylpyrocarbonate (DEP). The absorbance change accompanying the inactivation showed that a single histidyl residue was modified by DEP, resulting in a complete loss of enzymatic activity. In the presence of acetate, the enzyme was completely protected from DEP-inactivation. Furthermore, DEP-inactivated enzyme recovered its enzymatic activity on treatment with hydroxylamine. These observations seem to imply that the modified histidine is essential for enzyme activity. In addition, modification by DEP changed the absorption spectrum in the visible region. This strongly suggests that the modified histidyl residue is present in the vicinity of the flavin moiety of the enzyme molecule.
D055598:Chemical Phenomena; D002621:Chemistry; D003352:Corynebacterium / Q000201:enzymology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D008670:Metals / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050879:Sarcosine Oxidase; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D008670:Metals; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D006639:Histidine; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D004047:Diethyl Pyrocarbonate