Purification and molecular and catalytic properties of phosphoribulokinase from the cyanobacterium Chlorogloeopsis fritschii

Abstract

Phosphoribulokinase (ATP: D-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was purified from the cyanobacterium Chlorogloeopsis fritschii. The enzyme had a molecular weight of 230,000, as determined by gel filtration, and a pH optimum of 8.6. Divalent cations were essential for activity, maximal activity being supported by Mg2+, while Mn2+, Ca2+ and Co2+ were less effective. AMP, ADP, phosphoenolpyruvate, aspartate and malate inhibited enzyme activity completely at 1 mM. No effects on phosphoribulokinase activity were observed with NAD, NADH, NADP or NADPH at up to 10 mM or with glyoxylate at up to 20 mM. The enzyme was activated in semi-purified extracts by the addition of dithiothreitol and reduced glutathione. SDS-PAGE of SDS-dissociated enzyme revealed only one polypeptide band of molecular weight 40,000. This suggests that C. fritschii phosphoribulokinase is a hexamer consisting of six subunits of identical size.