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 P46) removes an approximately 15-kDa C-terminal domain generating a 30-kDa species (P30) which is stable against further digestion. While P30 is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P46 to the active 41-kDa form of GPR (P41). Trypsin treatment of P41 cleaves the same bond in the C-terminal part of the protein as is cleaved in the P46-->P30 conversion. While the approximately 29-kDa species generated by trypsin treatment of P41 is active, it is rapidly degraded further by trypsin to small inactive fragments. These results, as well as a thermal melting temperature for P41 which is 13 degreesC lower than that for P46 and the unfolding of P41 at significantly lower concentrations of guanidine hydrochloride than for P46, are further evidence for a difference in tertiary structure between P46 and P41, with P46 presumably having a more compact stable structure. However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P46 and P41. The removal of approximately 30% of P46 or P41 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 P41 by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.
Nessi
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Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
Jedrzejas
M J
MJ
Setlow
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D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology* / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012808:Sigma Factor; D013171:Spores, Bacterial / Q000201:enzymology; D014157:Transcription Factors; D014357:Trypsin
D001426:Bacterial Proteins; D008670:Metals; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C104642:spore germination protease