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.

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A mutant of Bacillus stearothermophilus deficient in pyruvate carboxylase (EC 6.4.1. l) is inhibited in its growth in a salts-acetate medium on the addition of lactate or glucose to the acetate culture. Secondary mutants isolated for ability to grow in a salts-acetate lactate medium are still deficient in pyruvate carboxylase but arecapable of synthesizing the glyoxylate-cycle enzyme, isocitrate lyase (EC 4.1.3.1), at an elevated rate (Sundaram,

Malate synthases from a thermophilic Bacillus and Escherichia coli have been isolated in a high state of purity. Molecular weights of these two proteins determined in the native state and after denaturation in sodium dodecyl sulfatemercaptoethanol show that the enzymes are monomeric. This conclusion is supported, for the thermophile enzyme, by the result ofan electrophoretic analysis of that protein after treatment with dimethylsuberimidate and denaturation. The thermophilic Bacillus malate synthase is considerably more thernostable than its mesophilic counterparts from E. coli, Bacillus licheniformis, and Pseudomonas indigofera. It is, however, markedly labilized by an increase in the ionic strength of the medium brought about by the addition of 0.2 M potassium chloride or in pH above 9. Increased ionic strength has little effect on the thermostability of the mesophilic bacterial malate synthases. These observations provide strong support for the idea that monomeric proteins in thermophiles owe their unusual heat stability to the presence of salt bridges in their tertiary structure.