Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp

Abstract

Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.

Isocitrate Lyase from Flax

“…Only in size do they differ appreciably. The bacterial enzymes are 206,000 and 180,000 daltons from Pseudomonas indigofera and a thermophilic Bacillus sp., respectively (6,29), and the green algal enzyme from Chlorellapyranoidosa has a mol wt of 180,000 (21), a value similar to that reported for lupine isocitrate lyase (43). In contrast, enzymes from Neurospora crassa, flax, and cucumber have mol wt of 270,000 (20), 264,000 (23), and 255,000 (14), respectively.…”

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme

“…Inhibition patterns and con- (Fig. 4) 48,000 has been determined (8,38), whereas the ICLs from Acinetobacter calcoaceticus and a Rhodopseudomonas species exhibited a subunit weight of around 63,000 (20,45 …”

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase

“…The specific activity of the purified enzyme is similar to that from E. coli (32) and a thermophilic Bacillus sp. (7). The Km of purified ICL for isocitrate was 40 ,uM.…”

“…The glyoxylate bypass circumvents the two carbon dioxide losing steps of the tricarboxylic acid cycle, allowing net gain of carbon into cellular structures. Even though ICL has been purified from several bacteria (7,22,32,35,37), it is not a well-studied enzyme. ICL from A. calcoaceticus B4 has been purified to homogeneity.…”