III. RESOLUTION OF PYRIDOXAL 5'-PHOSPHATE AND COENZYME SPECIFICITY*

SUMMARY o-Serine apodehydratase is much more labile to inactivation by oxidation and denaturation than the corresponding holoenzyme (EC 4.2.1.14). In the presence of dithiothreitol and acetylated serum albumin, which stabilize the apoenzyme at high dilutions, reversible dissociation of holoenzyme (20 no) to pyridoxal-P and apoenzyme occurred over several hours; both resolution and reactivation with pyridoxal-P are slow processes. The rate of resolution is greatly increased by L-cysteine, and less so by D-cysteine. In phosphate bufEer, L-cysteine reacts with free pyridoxal-P about 10 times faster than with D-serine holodehydratase. Inactivation (resolution) of the holoenzyme by r+-cysteine proceeds at the same rate as do spectral changes at 325 mn and at 415 nm; it is much faster in imidazole-citrate than in phosphate buffer, and is faster at pH ‘7.8 than at pH 6.8. 2-Norpyridoxal-P, 2’-methylpyridoxal-P, Z’-hydroxypyridoxal-P, 6-methylpyridoxal-P, 2-nor-6-methylpyridoxal-P, and 5’-methylpyridoxal-P combine with D-serine apodehydratase to yield active analogue-holoenzymes of varied catalytic efficiencies. 2’,2’-Dimethylpyridoxal-P, 2’-propylpyridoxal-P, pyridoxal 5’-methylphosphonate, pyridoxal 5’-(P-cyanoethylphosphate), and P-(2-methyl-3-hydroxy-4formylpyridine-5)-propionate combined with the apoenzyme, as shown by both spectral data and inhibition of reactivation by pyridoxal-P, but the complexes formed were catalytically inactive. N- Methylpyridoxal- P, 3 - 0 - methylpyridoxal- P, and 5-deoxypyridoxal neither activated nor inhibited, and failed to show spectral evidence of interaction with the apoenzyme. Thus a free phenolic group at position 3 and the unsubstituted heterocyclic nitrogen of pyridoxal-P contribute importantly to binding of this coenzyme; they may be required for catalytic activity as well. Alterations at positions 2 and 6 modify the affinity of the coenzyme analogues for the apoenzyme, but do not preclude their functioning in a catalytic capacity. All replacements of the 5’-phosphate group of pyridoxal-P led to analogues that were catalytically inert; however, since the geometry of binding may differ for such analogues, the result does not necessarily imply a catalytic role for the phosphate group.