Regulatory properties of citrate synthase from Rickettsia prowazekii.

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Abstract
Citrate synthase (citrate (si)-synthase); (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (R. prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative MW of .apprx. 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl CoA saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP > ADP .mchgt. AMP). [.beta.,.gamma.-methylene]ATP, dATP and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl CoA was present in high concentration (.gtoreq. 50 .mu.M). Neither NADH nor .alpha.-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eukaryotic and gram-positive prokaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the 1st characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.