Abstract
Leucine dehydrogenase (EC 1.4. 1.9) from Bacillus sphaericus was inactivated by pyridoxal 5'- phosphate, although less than 50% by one treatment. The inactivation was pH-dependent; the enzyme was most easily inactivated at pH 8.0. The inactivation was reversible by dialysis of the inactivated enzyme, but became irreversible when the inactivation was followed by reduction of the enzyme with NaBH4. The inactivation by pyridoxal 5'-phosphate was accompanied by the concomitant appearance of a peak at 427nm, which was shifted to 325nm by reduction with NaBH4. Amino acid analysis of an acid hydrolysate of the pyridoxal 5'-phosphate-inactivated and NaBH4-reduced enzyme showed the presence of ε-N-pyridoxyl-lysine. Thus, the inactivation is probably due to Schiff base formation between pyridoxal 5'-phosphate and an ε-amino group of a lysine residue of the enzyme. The enzyme was fully protected from inactivation only by NADH. The repeated treatment of the enzyme with pyridoxal 5'-phosphate and NaBH4 led to complete irreversible inactivation of the enzyme through a stepwise decrease in the activity. The substrate specificity and Km values of the partially inactivated enzyme were not essentially different from those of the native one.
