The role of bound calcium ions in thermostable, proteolytic enzymes. I. Studies on thermomycolase, the thermostable protease fron the fungus Malbranchea pulchella.

Thermomycolase, the thermostable, extracellular, serine protease of the fungus Malbranchea pulchella (G. Voordouw, G. M. Gaucher, and R. S. Roche (1974), Can J. Biochem. 52, 981-990), binds one calcium ion with an apparent binding constant of 5 X 10(5) M-1 at 25degreesC, pH 7.50, and ionic strength 0.1. There is very little change in the overall conformation of thermomycolase upon binding of this calcium ion: no change can be detected, beyond experimental error, in the sedimentation coefficient or the aromatic and peptide circular dichroism of the enzyme. However, binding of calcium changes the absorption spectrum, the ultraviolet difference spectrum being characterized by a strong band at 237 nm and smaller bands at 280 and 295 nm. The difference molar extinction coefficient at 237 nm parallels the calcium-binding isotherm. The small changes in equilibrium properties are constrasted by large calcium-dependent changes in the rates of autolytic degradation and thermal and urea denaturation. The dependence of the second-order rate constant for autolytic degradation on the free calcium ion concentration can be quantitatively accounted for by a model in which only conformers with an unoccupied calcium binding site serve as substrates in the reaction. The calcium dependence of the first-order rate constant for thermal denaturation at 70degreesC and pH 7.0 can also be accounted for quantitatively by a model in which the critically activated intermediate has a smaller calcium-binding constant than the native form of the enzyme under these conditions. The same model also accounts for the denaturation in 8 M urea at 50degreesC, pH 7.0. Rates of hydrogen-tritium exchange are shown to decrease when the calcium ion is bound. Irrespective of the occupancy of the calcium-binding site 33% of the backbone peptide hydrogens of thermomycolase do not exchange within 24 hr at 25degreesC, pH 8.0, and ionic strength 0.1.