^[1]Grimelysin (ECP 32), discovered, purified and initially characterized as protease ECP 32 [6,7,8], was later shown to be identical to grimelysin [18]. Therefore, the properties of the enzyme identified for ECP 32 could be applied to grimelysin. However, here we retain the name grimelysin (ECP 32) and ECP-cleaved actin to comply with the published data where the protease was named ECP 32. Grimelysin (ECP 32), purified from a bacterial extract using sequential chromatography steps, is a single 32 kDa polypeptide, whose N-terminal sequence was determined to be AKTSSAGVVIRDIFL [8]. The optimum of the protease activity was observed in the range of pH 7–8 when actin and melittin were used as substrates [8,23]. The proteolytic activity increased with increasing ionic strength: in 50–100 mM NaCl the activity of grimelysin (ECP 32) towards melittin was shown to be nearly twice higher than in a low ionic strength solution [23,24]. It was also enhanced in the presence of millimolar ATP concentrations, though hydrolysis of melittin was not accompanied by ATP hydrolysis at a rate comparable with the cleavage rate. This implies that protease grimelysin (ECP 32) is not an ATP-dependent enzyme [23], which is important for the experiments involving actin because actin contains ATP as a tightly-bound nucleotide. The protease activity is inhibited by EDTA, EGTA, o-phenanthroline and zincone, and the EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions [2,3]. Based on these data, grimelysin (ECP 32) was classified as a neutral metalloproteinase (EC 3.4.24) [8].

Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 [10] was observed at enzyme: substrate mass ratios of 1:25 to 1:3000 [8]. Two more sites, between Ala-29 and Val-30 and between Ser-33 and Ile-34, were cleaved by ECP 32 in heat- or EDTA-inactivated actin, apparently due to conformational changes around residues 28–34 buried in intact actin [8]. Besides actin, only melittin [18,19], histone H5, bacterial DNA-binding protein HU and chaperone DnaK [25] were found to be protease substrates. In agreement with this high substrate specificity, ECP 32 did not hydrolyze tropomyosin, troponin, α-actinin, casein, histone H2B, ovalbumin, bovine serum IgG, bovine serum albumin, bovine pancreatic ribonuclease A, trypsin, human heat shock protein HSP70, chicken egg lysozyme [7], insulin [24], DNAse I [9,13], gelsolin [14] and profilin [26]. The amino acid residues recognized by grimelysin (ECP 32) in actin and melittin are hydrophobic. This specificity is characteristic for thermolysin-like metalloproteinases [27]. However, high specificity of the enzyme seems to be determined predominantly by conformation at the actin cleavage site rather than its primary structure.

        Grimelysin was obtained as a recombinant protein. This has been achieved by cloning the putative gene encoding grimelysin in S. grimesii A2 and in the reference S. grimesii 30063 [18] using published protealysin sequences identified in S. proteamaculans [19]. Grimelysin shared all properties characteristic for ECP 32 including a molecular weight of 32 kDa, an N-terminal 14 amino acid sequence, optimum activity in the range of pH 7–8 and inhibition with o-phenanthroline and EGTA [18].

       Protealysin is a neutral zink-containing metalloprotease of Serratia proteamaculans. The protealysin gene was cloned from a genomic library of S. proteamaculans strain 94 isolated from spoiled meat. This protein was expressed in Escherichia coli and purified as described earlier [19]. Similarly to other thermolysin-like proteases [27,28], protealysin is synthesized as a precursor containing a propeptide of about 50 amino acids that is removed during formation of mature active protein [29]. The propeptide is much shorter than the propeptides of the thermolysin-like proteases and has no significant structural similarity to the propeptides of most thermolysin-like proteases [30,31,32]. A similar propeptide of 50 amino acids was also detected in the primary structure of the recombinant grimelysin. According to SDS-electrophoresis, recombinant proteins with or without propeptide had an apparent molecular weight of 37 and 32 kDa, respectively [19].

 The molecular weight of the active recombinant protealysin 32 kDa and the N-terminal amino acid sequence AKTSTGGEVI are identical to those of grimelysin [8,19]. The optimal pH for azocasein hydrolysis is 7, and protealysin is completely inhibited by o-phenanthroline [19], i.e., has the same properties as grimelysin [8,18]. Protealysin and grimelysin (ECP 32) are also similar in their unique property of being able to digest actin specifically [8,9,10,22,33].

 

 

 

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