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

@entity141 @entity13667 @entity291 @entity460
@entity460 -dependent protease II ( @entity13667 -II) from @entity291 heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM @entity141 for half-maximal activity and that the large subunit contains both the catalytic and @entity141 binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of @entity141 on structural and catalytic properties of @entity13667 -II in the presence and absence of substrate proteins. When incubated with @entity141 in the absence of substrate, @entity13667 -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 @entity141 (half-maximal rate at approximately 600 microM @entity141 ). The first cleavage appears to be unimolecular because its rate is unaffected by @entity13667 -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 @entity13667 -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 results indicate that the 80-kDa/26-kDa form of @entity13667 -II represents an inactive proenzyme and that the initial @entity141 -dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a @entity141 concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the @entity141 concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of @entity13667 -II activity.
