Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C. sphenoides.
Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate. The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the L-glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration. Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate.
D001419:Bacteria / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D003013:Clostridium / Q000201:enzymology; D005971:Glutamates / Q000502:physiology*; D018698:Glutamic Acid; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D012241:Rhodopseudomonas / Q000201:enzymology; D013291:Streptococcus / Q000201:enzymology
D005971:Glutamates; D009097:Multienzyme Complexes; D018698:Glutamic Acid; D007652:Oxo-Acid-Lyases; C019909:citrate (pro-3S)-lyase