Amylase activity and stability at high and low temperature depending on calcium and other divalent cations

Since the extracellular amylase from B. caldolyticus had been characterized as a Ca-dependent enzyme, we wanted to determine the function and specificity of this ion, and it's possible significance for the thermophilic properties of the enzyme. The first question concerned the substitution of Ca with regard to the activity at 70 C. Our data show that Sr or other divalent cations can only to a limited extent replace Ca, and that only in presence of Ca full activity can be achieved. When the majority of the binding sites of the subunits are occupied by cations other than Ca, an almost total loss of activity results. The reason for this kind of inactivation was found in the responsibility of Ca for the stability of the enzyme at high temperature. The omission of Ca then leads to an irreversible denaturation of the enzyme, so that after this kind of treatment no activity is detectable at either high or low temperature, which means that the amylase is not a thermostable enzyme in the classical sense. The stabilizing effect of Ca could not be substituted by any other of the cations tested. Experiments concerning the stability of the enzyme at various temperatures in absence of presence of Ca revealed that the enzyme is stable up to 55 C as long as trace amounts of Ca are available. If these are omitted by a chelator, the enzyme becomes unstable between 40 and 45 C. Experiments in which a certain protein concentration range was tested at 40 and 70 C with given concentrations of Ca showed that the stability, and with it the activity, at high temperature is directly related to the amount of Ca available: The more the Ca supply is limited, the less enzyme protein can be kept in the right configuration, and irreversibly denatures as a result. At low temperature, however, the enzyme becomes almost independent of Ca, and the small amounts necessary to obtain full activity can be replaced by other divalent cations. The main conclusions concerning the role of Ca are that at high temperatures it participates in achieving the correct configuration of the thermostable form of the enzyme, and that in this function it is irreplacable. At low temperature it acts as an unspecific cofactor. The other conclusion is that the amylase can exist either as a caldo-active, thermostable, Ca-dependent enzyme, or in a Ca-independent thermostable state with a lower activity.