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Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.