Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius

Abstract

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.

Enzymatic Activity of the Staphylococcus aureus SplB Serine Protease is Induced by Substrates Containing the Sequence Trp-Glu-Leu-Gln

“…According to mutagenesis studies, histidine of the S1 subsite substantially contributes to precise substrate positioning. 42,43 In comparison with S1, other subsites in chymotrypsin-like proteases are generally not that very well defined on the structural level. However, enough information is available to suggest likely S2-S4 SplB subsites and substrate positioning.…”

Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

“…H184 of GluV8 is located close to S169(Figure 4) and highly conserved among endopeptidases from staphylococci(Carmona and Gray, 1987;Ohara-Nemoto et al, 2002; Yokoi et al, 2001), Bacillus species(Kakudo et al, 1992;Svendsen and Breddam, 1992;Rebrikov et al, 1999) and Streptomyces species(Suzuki et al, 1994;Svendsen et al, 1991), indicating its importance. In fact, the substitution of H186T of B. intermedius endopeptidase (equivalent to H184 of GluV8) resulted in a 615-fold reduction in k cat , but had little effect on the K m (4.9-fold increase)(Demidyuk et al, 2004).…”

The novel ADAMs-like microbial metalloendopeptidase

“…Proteolytic activity of metalloendopeptidase was determined by azocasein cleavage [27,28]. Measure ments were made at a wave length of 450 nm.…”