title: 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.
authors.affiliation: Laboratory of Protein Engineering, Institute of Molecular Genetics of Russian Academy of Sciences, Moscow 123182. duk@img.ras.ru
authors.forename: I V
authors.initials: IV
authors.lastname: Demidyuk
authors.forename: D V
authors.initials: DV
authors.lastname: Romanova
authors.forename: E A
authors.initials: EA
authors.lastname: Nosovskaya
authors.forename: G G
authors.initials: GG
authors.lastname: Chestukhina
authors.forename: I P
authors.initials: IP
authors.lastname: Kuranova
authors.forename: S V
authors.initials: SV
authors.lastname: Kostrov
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D009154:Mutation; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics / Q000502:physiology
chemical_list: D007328:Insulin; D006639:Histidine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase