Biphasic inactivation of procine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.
Temperature studies have indicated that from 0 to 37 degrees, the time-dependent inactivation of mitochondrial malate dehydrogenase from porcine heart by pyridoxal 5-phosphate (pyridoxal-5-P) is biphasic. The initial phase of the inactivation is reversible but can be made irreversible by reduction with sodium borohydride. The reduced pryidoxal-5-P-enzyme adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325. The irreversible second phase of the inactivation is accompanied by the appearance of a new 325-nm absorbance maximum, in the absence of reduction, and a fluorescence emission centered about 390 to 400 nm when excited at 325. The evidence presented suggests the formation of a Schiff base between pyridoxal-5-P and a nucleophilic residue, most likely lysine, of malate dehydrogenase during the first phase of inactivation. An X-azolidine-like structure, a further derivative of the Schiff base, possessing spectral properties consistent with the reported data, may be formed during the second phase; this presumably involves a second nucleophilic residue of the enzyme, implicating the action of pyridoxal-5-P as a bifunctional reagent in this instance. The presence of the coenzyme, NADH, protects the enzyme from inactivation, suggesting that pyridoxal-5-P interacts at or near the malate dehydrogenase active center. Simultaneous binding studies using pyridoxal-5-P with known malate dehydrogenase competitive inhibitors AMP, ADP, and nicotinamide indicate that the pyridoxal-5-P modification occurs in the general area of the ADP portion of the coenzyme binging site. Furthermore, the presence of nicotinamide enhances pyridoxal-5-P binding to and inactivation of malate dehydrogenase.
D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors*; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013696:Temperature
D011732:Pyridoxal Phosphate; D008291:Malate Dehydrogenase