Mode of glucan degradation by purified phosphorylase forms from spinach leaves

The glucan specifity of the purified chloroplast and non-chloroplast forms of -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69–75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization 11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins G₅–G₁₁), the chloroplast phosphorylase had a 7–10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length required for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (K _i-0.1 mmol 1^-1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of maltodextrin phosphorylase.Abbreviations G1P Glucose 1-phosphate - MES 2(N-morpholino)ethane sulphonic acid - P_i orthophosphate - Tris Tris(hydroxymethyl)aminomethane