Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal.
Pyridoxal phosphate reacts with not only the lysyl residue(s) essential for enzymatic activity but also other reactive lysyl residues in rabbit muscle lactate dehydrogenase (EC 1.1.1.27). To raise the specificity of pyridoxal phosphate, adenosine diphospho-, triphospho-, and tetraphosphopyridoxals have been newly synthesized and used for modification of the enzyme. Incubation of the enzyme for 30 min with the diphospho, triphospho, and tetraphospho compounds all at 1 mM followed by reduction by sodium borohydride resulted in the loss of enzymatic activity by 64, 51, and 34%, respectively. NADH almost completely protected the enzyme from inactivation, whereas pyruvate showed no protection. Binding of the reagents to the enzyme subunit in an equimolar amount corresponds to the complete inactivation. The adenosine diphosphopyridoxal modified enzymes with different residual activities were chromatographed on a Blue Toyopearl affinity column. The results showed the presence of at least four enzyme species besides the intact enzyme that are significantly different from one another in the amount of the reagent bound, the affinity for NADH, and the specific activity. The decrease in the affinity of the enzyme for NADH and the loss of enzymatic activity paralleled in the modification by adenosine diphosphopyridoxal, whereas, in the modification by pyridoxal phosphate, the decrease in the affinity for NADH preceded the inactivation. It is concluded that modification by adenosine polyphosphopyridoxal compounds are specific for the active site lysyl residue(s) in lactate dehydrogenase.
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D011730:Pyridoxal / Q000031:analogs & derivatives*; D011732:Pyridoxal Phosphate / Q000494:pharmacology* / Q000031:analogs & derivatives; D011817:Rabbits
D000227:Adenine Nucleotides; D007202:Indicators and Reagents; C049517:adenosine triphosphopyridoxal; C049518:adenosine tetraphosphopyridoxal; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; C048236:pyridoxal 5'-diphospho-5'-adenosine