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 Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM 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.
Jaklitsch
W M
WM
Department of Biochemistry, King's College London, Kensington, U.K.
Kubicek
C P
CP
D001667:Binding, Competitive; D002413:Cations, Divalent; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004228:Dithionitrobenzoic Acid; D004355:Drug Stability; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000494:pharmacology*; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology*; D010408:Penicillium chrysogenum / Q000201:enzymology*
D002413:Cations, Divalent; D007527:Isoenzymes; D007656:Ketoglutaric Acids; D009711:Nucleotides; D004228:Dithionitrobenzoic Acid; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine