CITRATE SYNTHASE FROM THE OBLIGATE METHYLOTROPH METHYLOBACILLUS-FLAGELLATUM

Citrate synthase (CS; E.C. 4.1.3.7) was purified from an obligate meth ylotroph Methylobacillus flagellatum using ammonium sulfate precipitat ion, gel-filtration, ion-exchange chromatography, hydroxylapatite chro matography and Blue-Sepharose CL-6B chromatography. The M(r) of the na tive enzyme was estimated to be 350000 (+/-5000). The enzyme consists of eight identical subunits with an M(r) of 46 000 (+/-2000) (SDS-PAGE ) and has a pH optimum of 9.0. Michaelis-Menten kinetics were observed for the reaction catalyzed by the citrate synthase. The apparent K(m) values were 36.5 muM for acetyl-CoA and 20.8 muM for oxaloacetate. Th e enzyme was insensitive to the influence of intermediate metabolites with the exception of ATP at 5 mM, which caused 55% inhibition of acti vity.