THE REDUCTIVE TRICARBOXYLIC-ACID CYCLE OF CARBON-DIOXIDE ASSIMILATION- INITIAL STUDIES AND PURIFICATION OF ATP-CITRATE LYASE FROM THE GREEN SULFUR BACTERIUM CHLOROBIUM-TEPIDUM

Carbon dioxide is fixed largely by the reductive tricarboxylic acid (R TCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citra te lyase, was purified to apparent homogeneity from the moderately the rmophilic green sulfur bacterium Chlorobium tepidum. The molecular wei ght of the native enzyme,vas about 550,000, and the preponderance of e vidence indicated that the protein is composed of identical subunits ( M-r of congruent to 135,000) which degraded to two major proteins with M(r)s of congruent to 65,000 and congruent to 42,000. Western immunob lots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous pr otease, similar to what has been observed with mammalian, yeast, and m old ATP-citrate lyase. In addition to apparent structural similarities , the catalytic properties of C. tepidum ATP-citrate lyase showed mark ed similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C . tepidum ATP citrate lyase occurred, presumably on a histidine residu e at the active site, similar to the ease for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and G TP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system shou ld serve as an excellent model for studies of the enzymology and regul ation of this protein.