WATER-INSOLUBLE ENZYMES - KINETICS OF RABBIT MUSCLE ENOLASE EMBEDDED WITHIN AN INSOLUBLE CARRIER

Crystalline rabbit muscle enolase was transformed into a water-insoluble form by embedding the enzyme in a highly cross-linked, synthetic polymer, without formation of covalent bonds between enzyme and insoluble carrier, according to the procedure previously published by us (Science 142, 678 (1963); Arch. Biochem. Biophys. 127, 779 (1968)). Enzyme kineties of the insoluble form of enolasc were compared to those of the soluble form. There were no differences between the two forms of enolasc with regard to the influence of variations in pH, enzyme concentration or substrate concentration on enzyme activity. Michaelis constants, maximum rates of substrate turnover and degrees of substrate inhibition were the same for both forms of the enzyme. This behavior of insoluble enolase is consistent with that of an enzyme immobilized in an electrostatically neutral, insoluble carrier. Considerable differences existed between the two forms of enolase in their behavior toward temperature and magnesium. Between 1 and 24 °, the activity of the insoluble form increased much more slowly than that of the soluble form; optimum activity was reached at 44 and 50 ° for the soluble and insoluble forms, respectively; the former was no longer active at 50 °, but insoluble enolase still retained 23% of its activity at 56 °. Activation energies were similar for both forms of the enzyme in the upper temperature range (above 24 °); i.e., 11,800 and 15,300 cal/mole for the soluble and insoluble forms, respectively. But they differed widely at lower temperatures (below 8 °); i.e., 29,600 and 4600 cal/mole, respectively. Both forms of enolase required magnesium for maximum activity (0.68 × 10-3 m), but the insoluble form, in contrast to soluble muscle enolase, was not inhibited by an excess of magnesium. Zinc inhibited both forms of the enzyme to about the same extent, except at low magnesium concentrations where the insoluble form was some-what less affected by Zn2+ than soluble enolase. © 1969.