Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids.
The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of beta-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.
de Kreij
A
A
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Venema
G
G
van den Burg
B
B
D000596:Amino Acids / Q000737:chemistry*; D051792:Basic Helix-Loop-Helix Proteins; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D029721:Drosophila Proteins; D010450:Endopeptidases / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010802:Phylogeny; D017433:Protein Structure, Secondary; D012097:Repressor Proteins; D013379:Substrate Specificity; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D051792:Basic Helix-Loop-Helix Proteins; D002364:Caseins; D029721:Drosophila Proteins; D010450:Endopeptidases; D019476:Insect Proteins; D008666:Metalloendopeptidases; D010455:Peptides; D012097:Repressor Proteins; D013820:Thermolysin; C121961:E(spl)m4-BFM protein, Drosophila; C044194:microbial metalloproteinases