OBSERVATIONS ON THE FUNCTION OF PYRIDOXAL-5-PHOSPHATE IN PHOSPHORYLASE

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Abstract
Various experiments performed to elucidate the mechanism of the phosphorylase reaction have failed to demonstrate any direct participation of the phosphate group of pyridoxal-5-phosphate in the enzymatic reaction. Improved methods were developed for the resolution of phosphorylases a and b. One of the methods led to the crystallization of apophosphorylase a. Apophosphorylase b was converted to apophosphorylase a with phosphorylase kinase and adenosine triphosphate. The apophosphorylases exist in solution as mixtures of molecular species corresponding in size to a basic monomer unit, about 125,000 molecular weight, or multiples of it. Recombination with pyridoxal-5-phosphate, pyridoxal, or 5-deoxypyridoxal causes the protein to resume its original molecular size. The latter two analogs do not restore enzymatic activity but [omega]-methylpyridoxal-5-phosphate can reactivate to about 50%. Pyridoxal and 5-deoxypyridoxal can combine with apophosphorylase a as shown by the fact that preincubation with these substances inhibits subsequent reaction of the protein with pyridoxal-5-phosphate. Combination of the apoenzyme with pyridoxal-5-phosphate can be prevented by preincubation of the latter compound with isonicotinic acid hydrazide, but no inhibition of the enzymatic activity is observed when phosphorylase a is incubated with this reagent. These findings prove that, although the combination of apophosphorylase a with pyridoxal-5-phosphate is through the 4-formyl group of the latter, no Schiff-base linkage exists in phosphorylase. It is concluded that the formation of a Schiff base between pyridoxal-5-phosphate and protein amino groups is the rate-limiting, intermediate reaction and is followed by further rearrangement to the stable bond structure in phosphorylase.