Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione.
The inactivation of cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart and the specific modification of arginyl residues have been found to occur when the enzyme is inhibited with the reagent butanedione in sodium borate buffer. The inactivation of the enzyme was found to follow pseudo-first order kinetics. This loss of enzymatic activity was concomitant with the modification of 4 arginyl residues per molecule of enzyme. All 4 residues could be made inaccessible to modification when a malate dehydrogenase-NADH-hydroxymalonate ternary complex was formed. Only 2 of the residues were protected by NADH alone and appear to be essential. Studies of the butanedione inactivation in sodium phosphate buffer and of reactivation of enzymatic activity, upon the removal of excess butanedione and borate, support the role of borate ion stabilization in the inactivation mechanism previously reported by Riordan (Riordan, J.F. (1970) Fed. Proc. 29, Abstr. 462; Riordan, J.F. (1973) Biochemistry 12, 3915-3923). Protection from inactivation was also provided by the competitive inhibitor AMP, while nicotinamide exhibited no effect. Such results suggest that the AMP moiety of the NADH molecule is of major importance in the ability of NADH to protect the enzyme. When fluorescence titrations were used to monitor the ability of cytoplasmic malate dehydrogenase to form a binary complex with NADH and to form a ternary complex with NADH and hydroxymalonate, only the formation of ternary complex seemed to be effected by arginine modification.
D000249:Adenosine Monophosphate; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002074:Butanones; D003593:Cytoplasm / Q000201:enzymology*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008314:Malonates / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding
D002074:Butanones; D008314:Malonates; D000249:Adenosine Monophosphate; D001120:Arginine; D008291:Malate Dehydrogenase