Mutants of Bacillus stearothermophilus de-repressed for isocitrate lyase and malate synthase.

philes Pseudomonas indigofera (Shiio et al., 1965) and Chlorella pyrenoidosa (John 8c Syrett, 1967) is apparently similar. In mutant PC2 NG35 cells the lyase accounts for 16 % of the soluble proteins. The corresponding proportion of malate synthase is 2.5% in the wild-type and 6% in the mutant. Themolecularweightsof thenativeenzymes, determined by gelfiltrationonaSepharose 6B column, are given in Table 2. The sedimentation and diffusion behaviour of the enzymes in the analytical ultracentrifuge was consistent with these values. Table 2 also presents the molecular weights of the subunits of the enzymes, data on several kinetic parameters and thermostability of the two enzymes and some comparative data on isocitrate lyases from Ps. indigofera and C. pyrenoidosa. The thermophilic lyase, like the Ps. indigofera enzyme, is apparently a tetramer and generally resembles the two mesophilic cases in molecular size and kinetic parameters. Its greater thermostability, lower turnover number and salt-activation property distinguish it from its mesophilic counterparts. The salt activation must be an intrinsic characteristic of the thermophilic enzyme molecule rather than being due to extraneous factors, since the homogeneous enzyme manifests this property. We have now observed that isocitrate lyases from two more mesophiles, Ps. indigofera and Neurospora crassa, are not activated by salt. The thermophilic malate synthase appears to be a monomer, as judged by the molecular weights of the native enzyme and of its subunit, unless it is considered possible that the interaction between subunits in the enzyme molecule is strong enough to withstand the denaturing conditions in our experiments. The enzyme from yeast, which, to our knowledge, is the only mesophilic malate synthase to be reasonably well characterized, is an oligomer (Schmid et al., 1974). The relatively great thermostability of the thermophilic synthase may derive, at least in part, from its monomeric constitution.