Purification and characterisation of an α-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus

Abstract

An α-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. T. thermophilus phosphorylases hares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M yridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of T. thermophilus phosphorylase was observed at 70°C in the pH range 5.5-65. While at 25°C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70°C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other α-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (V(max) = 18 U/mg). Correspondingly, maltotriose functions as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T. thermophilus phosphorylase differs from that of known α-glucan phosphorylases.