Modification of pig M4 lactate dehydrogenase by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue.
1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.
D000818:Animals; D001665:Binding Sites; D001894:Borohydrides; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008239:Lysine / Q000032:analysis; D009132:Muscles / Q000201:enzymology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013552:Swine
D001894:Borohydrides; D007527:Isoenzymes; D011732:Pyridoxal Phosphate; D007770:L-Lactate Dehydrogenase; D008239:Lysine