Structure and Mechanism of Action of the Protease That Degrades Small, Acid-Soluble Spore Proteins during Germination of Spores of Bacillus Species

The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination. Trypsin treatment of the 46-kDa GPR zymogen (termed P(46)) removes an ∼15-kDa C-terminal domain generating a 30-kDa species (P(30)) which is stable against further digestion. While P(30) is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P(46) to the active 41-kDa form of GPR (P(41)). Trypsin treatment of P(41) cleaves the same bond in the C-terminal part of the protein as is cleaved in the P(46)→P(30) conversion. While the ∼29-kDa species generated by trypsin treatment of P(41) is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P(41) which is 13°C lower than that for P(46) and the unfolding of P(41) at significantly lower concentrations of guanidine hydrochloride than for P(46), are further evidence for a difference in tertiary structure between P(46) and P(41), with P(46) presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P(46) and P(41). The removal of ∼30% of P(46) or P(41) without significant loss in enzyme activity localized GPR’s catalytic residues to the N-terminal two-thirds of the molecule. This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P(41) by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.