A. De Kreij et al., Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids, J BIOL CHEM, 275(40), 2000, pp. 31115-31120
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 ch
aracterized with an identical substrate set and a uniform set of assay cond
itions. Characterization with peptide substrates as well as high performanc
e liquid chromatography analysis of p-casein digests shows that the M4 fami
ly is a homogeneous family in terms of catalysis, even though there is a si
gnificant degree of amino acid sequence variation. The results of this stud
y show that differences in substrate specificity within the M4 family do no
t correlate with overall sequence differences but depend on a small number
of identifiable amino acids. Indeed, molecular modeling followed by site-di
rected mutagenesis of one of the substrate binding pocket residues of the t
hermolysin-like proteases of Bacillus stearothermophilus converted the cata
lytic characteristics of this variant into that of thermolysin.