title: Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.
abstract: The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: O R
authors.initials: OR
authors.lastname: Veltman
authors.forename: V G
authors.initials: VG
authors.lastname: Eijsink
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: A
authors.initials: A
authors.lastname: de Kreij
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: B
authors.initials: B
authors.lastname: Van den Burg
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005998:Glycine / Q000235:genetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D000409:Alanine; D005998:Glycine