The reactions of pyridoxal 5′-phosphate with the M 4 and H 4 isoenzymes of pig lactate dehydrogenase

Abstract

The H 4 and M 4 isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5′-phosphate. In the early stages, inactivation is largely reversible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5′-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Neither pyruvate nor lactate alone gives any protection. With the M 4 isoenzyme, complete protection against inactivation by pyridoxal 5′-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H 4 isoenzyme. In the course of irreversible inactivation of H 4 lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to resolve earlier controversy.