Atypical reaction of 'essential' sulfhydryl groups of malic enzyme with 2-nitro-5-thiocyanobenzoate and 2,4-dinitrophenylthiocyanate.
Under protective conditions N-ethylmaleimide irreversibly blocks most of the nonessential SH groups of pigeon liver malic enzyme (EC 1.1.1.40) leaving the oxidative decarboxylase activity intact. Reaction between the resultant prereacted enzyme and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to the modification of about seven SH residues/tetramer, of which four fast-reacting groups constitute the 'essential' groups responsible for the loss of oxidative decarboxylase activity. 2-Nitro-5-thiocyanobenzoate (NTCB) reacts atypically with the prereacted enzyme by substituting the four 'essential' SH residues with one cyano residue and three 2-nitro-5-thiobenzoate residues. The resulting enzyme derivative is 90% inactive. The cyanoenzyme derivative produced by cyanolysis of DTNB-modified prereacted enzyme or NTCB-modified prereacted enzyme has all four 'essential' SH groups substituted with cyano groups and possesses half of the original activity. Modification of prereacted enzyme by 2,4-dinitrophenylthiocyanate (DNPT), in contrast to NTCB, results in unequal substitution of the 'essential' residues with cyano residues and a single 2,4-dinitrophenyl residue. Dithiothreitol reactivates the DNPT-modified prereacted enzyme by regenerating three 'essential' SH residues, but failed to release the dinitrophenyl residue. The atypical reactions of prereacted enzyme with NTCB or DNPT require that the native conformation of the enzyme be retained, since these reagents react by substituting the SH groups of urea-denatured enzyme with only cyano groups.
D000818:Animals; D010856:Columbidae; D004136:Dinitrobenzenes / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000378:metabolism*; D009578:Nitrobenzenes / Q000494:pharmacology*; D011190:Potassium Cyanide / Q000494:pharmacology; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013861:Thiocyanates / Q000494:pharmacology*
D004136:Dinitrobenzenes; D009578:Nitrobenzenes; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; D008291:Malate Dehydrogenase; C010936:2,4-dinitrothiocyanatobenzene; D011190:Potassium Cyanide; C008658:2-nitro-5-thiocyanobenzoic acid; D004229:Dithiothreitol