Modification of essential arginine residues of pigeon liver malic enzyme.
The reaction of pigeon liver malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with dicarbonyl compounds (2,3-butanedione, methylglyoxal, 2,4-pentanedione, and phenylglyoxal) resulted in a rapid loss of its enzymatic activity. The inactivation showed pseudo-first-order kinetics for all the dicarbonyls studied. All the log (pseudo-first-order rate constants) vs. log (dicarbonyl concentration) plots had slopes of near one, indicating approx. 1 : 1 reagent-active site complexes. Butanedione inactivation was reversible and was buffer-dependent. Pentanedione-modified enzyme showed a new absorption peak at 310 nM. NADP could completely protect the enzyme from inactivation. Oxaloacetate, ADP, AMP, NMN and adenosine were also effective in protection. Complete inactivation of the enzyme was accompanied by a loss of about six arginine residues per enzyme monomer. Butanedione-modified enzyme still bound NADPH as shown by fluorescence titration, nor was it binding with NADP impaired as determined by equilibrium gel filtration. The arginine residues, therefore, do not function in the coenzyme binding. However, the binding between the modified enzyme and [14C]malate was significantly decreased. These results led us to conclude that the arginine residues of malic enzyme are involved in the binding of the carboxyl group of substrate malate.
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D010856:Columbidae / Q000378:metabolism*; D003931:Diacetyl / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D010422:Pentanones / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology; D011485:Protein Binding; D011765:Pyruvaldehyde / Q000494:pharmacology
D010422:Pentanones; D009249:NADP; D011765:Pyruvaldehyde; D001120:Arginine; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D003931:Diacetyl; C013186:2,3-pentanedione; D010658:Phenylglyoxal