PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

Abstract

ABSTRACTA single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced‐min‐1.mg‐1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N‐ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo‐tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg2+. The monovalent cation K+ acts as a strong activator. The Km values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with Vmax for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5‐bisphosphate, adenosine monophosphate (AMP), ribose‐5‐phosphate, and glyceraldehyde‐3‐phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3‐diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).