Comparative Glucan Specificities of Two Types of Spinach Leaf Phosphorylase

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Abstract
Two types of α-glucan phosphorylase [EC 2.4.1.1] from spinach leaves have been separately purified to near homogeneity. Type I enzyme shows a subunit molecular weight of 92,000 and K m values for amylopectin, glycogen and amylose much smaller than that for maltopentaose. Cyclodextrin is a normal competitive inhibitor with respect to maltopentaose, while it is a multi-site competitive type inhibitor with respect to amylopectin, glycogen or amylose. Type II enzyme shows a subunit molecular weight of 108,000, and utilizes amylopectin, amylose and maltopentaose well, but glycogen very poorly. On affinity electrophoresis, Type II enzyme shows no affinity for glycogen and the dissociation constant for amylopectin is more than a thousand-fold greater than that of Type I enzyme. Type II enzyme has similar characteristics (subunit size, glucan specificity, and mode of cyclodextrin inhibition) to potato phosphorylase, for which cyclodextrin is a normal competitive inhibitor with respect to either maltopentaose or amylopectin. Enzyme-glucan binding models have been proposed to explain these different kinetic properties. In spinach Type I phosphorylase, multiple glucan binding sites are located on the same face of the enzyme molecule to enable a single large substrate to be bound by riding on all the site; in spinach Type II and potato phosphorylases, two glucan binding sites are three-dimensionally arranged in a manner that excludes the possibility of the binding of amylopectin by riding on the two sites.