Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability.

Abstract

The total kinetic thermal stability of a protein molecule, expressed as the total free energy of activation in thermal denaturation reactions, can be separated into an intrinsic contribution of the polypeptide chain and a contribution due to the binding of calcium ions. The theory for this procedure is applied to thermal denaturation data, obtained at the pH of optimum stability, for the serine proteases, thermomycolase and subtilisin types Carlsberg and BPN', and for the zinc metalloendopeptidases, thermolysin and neutral protease A. The results, obtained from Arrhenius plots at high and low free calcium ion concentrations, reveal a considerable variation in the calcium ion contribution to the total kinetic thermal stability of the various enzymes. In the serine protease group, at 70 degrees C, the stability is largest for thermomycolase, mainly due to a relatively high intrinsic contribution. For the metalloendopeptidases the total kinetic thermal stability is largest for thermolysin, the difference between thermolysin and neutral protease A being dominated by bound calcium ion contributions. The intrinsic kinetic thermal stability of the polypeptide chain of thermolysin is considerably smaller than that of any of the serine proteases and is probably of the same order of magnitude as that of neutral protease A. Thus, the well known total kinetic thermal stability of thermolysin is due mainly to a single calcium ion (Voordouw, G., and Roche, R. S. (1975), Biochemistry 14, 4667) that binds with high affinity even at very high temperatures (K congruent to 6 X 10(7) M-1 at 80 degrees C).