Substratebinding Pockets

In the active site of PDF proteins, three substrate-binding pockets exist along with the metal-binding site. Using standard metalloprotease nomenclature,

Fig. 3.3 Schematic representation of the PDF active site and substrate transition state. The P1' substituent is represented as methionine.

these pockets are referred to as the S1', S2' and S3' pockets, and the corresponding positions on substrates or inhibitors are referred to as P1', P2' and P3' (Figure 3.3).

S1' pocket

The S1' pocket is the binding site for the methionine side chain of the substrate, and hence it is composed of mostly hydrophobic residues. This pocket remains extremely similar in size and shape among PDFs from various Gram-positive and Gram-negative organisms, with only subtle differences noted, such as the observation that S. aureus and S. pneumoniae PDFs possess a slightly wider (~1.4 A) pocket than H. influenzae and E. coli PDFs [46]. The only non-conservative amino acid residue substitution in this region lies on the border between the S1' and S3' pockets. This residue is a leucine in E. coli (Leu-125), H. influenzae (Leu-125), P. aeruginosa (Leu-127) and B. stearothermophilus (Leu-146), and a tyrosine in S. pneumoniae (Tyr-166), S. aureus (Tyr-147) and T. maritima (Tyr-122) [22, 46, 48]. This difference, lying on the solvent exposed edge of the pocket, seems to impart only subtle effects on binding or access to the pocket, as good enzyme inhibition can be obtained with inhibitors that bind only to the metal and into the S1 ' pocket in both Gram-positive and Gram-negative PDFs [46].

S2' pocket

The S2' pocket is not a true 'pocket', but rather an open, tunnel-like space pointing out into solvent. This loosely defined region is therefore able to accommodate a wide variety of side chains presented by the various formylated substrates to be processed by the PDF protein [63]. There is less overall conservation among the residues lining this region [46]. Type I PDFs generally possess a larger S2' site, suggesting that substrates with large substituents at P2' may be cleaved selectively by this class of enzymes [22].

S3' pocket

The S3' pocket is the least conserved region of the active site between Gramnegative and Gram-positive PDFs, due to a two residue insertion between the S1' and S2' regions in Gram-positive species (Gly-124 and Glu-125 in S. pneumoniae). Again, similar to the S2' pocket, this area is solvent-exposed and more of a surface depression than a true binding 'pocket' [46]. Thus, inhibitors that target specific residues within the S3' pocket are likely to be more selective for those particular PDFs.

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