HOMOCITRATE SYNTHASE FROM PENICILLIUM-CHRYSOGENUM - LOCALIZATION, PURIFICATION OF THE CYTOSOLIC ISOENZYME, AND SENSITIVITY TO LYSINE

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 ± 10 kDa as determined by gel filtration and a pH of 4.9 ± 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with K(m) = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 μM and h = 2.6. The enzyme was inhibited strongly by L-lysine (K(i) = 8 ± 2 μM; 50% inhibition by 53 μM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.