Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity

Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 μM Ca²⁺ for half-maximal activity and that the large subunit contains both the catalytic and Ca²⁺ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca²⁺ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca²⁺ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca²⁺ (half-maximal rate at approximately 600 μM Ca²⁺). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our result indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca²⁺-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca²⁺ concentration requirement of 350 μM for half-maximal rates. The further autoproteolysis, which results in the formation of the 8-kDa/17-kDa heterodimer, serves to reduce the Ca²⁺ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.