THE MECHANISM OF THE DE NOVO SYNTHESIS OF POLYSACCHARIDE BY PHOSPHORYLASE

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Abstract
In the de novo synthesis of amylose by phosphorylase early intermediates of chain growth consisting of 2 to 6 glucose units attached to the enzyme have been demonstrated. Under optimal conditions the rate of synthesis was about one amylose chain per phosphorylase monomer (mol. wt. 125,000) per 6 hours. De novo synthesis has been shown to proceed at its slow initial rate even in the presence of a-amylase. In this case the products are small amounts of amylortriaose, maltose, and glucose. A smaller extent of de novo synthesis of maltose, amylotriaose, and amylotetraose can be shown in a system where the initial concentration of Pi is 0.01 M and the ratio of Pi to G-1-P is 8.6. Apo-phosphorylase a (with pyridoxal-5[image]-phosphate removed) is inactive in de novo synthesis. When this protein is mixed with the native enzyme, the inactivated enzyme apparently can be used as the site of attach-ment of the de novo chains. When native phosphorylase a is treated with NaBH4 so that the bond linking pyridoxal-5[image]-phosphate to the enzyme becomes reduced, the enzyme still is active in de novo synthesis. These findings suggest that the attachment of the de novo amylose chain is to the enzyme protein and not to one of the functional groups of the coenzyme. Although in de novo synthesis enzyme-bound glucose presumably is an intermediate product, there is no evidence that a glucosylenzyme is an intermediate in the conventional phosphorylase reaction.

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