D. Bromme et al., ENGINEERING THE S-2 SUBSITE SPECIFICITY OF HUMAN CATHEPSIN-S TO A CATHEPSIN-L-LIKE AND CATHEPSIN-B-LIKE SPECIFICITY, The Journal of biological chemistry, 269(48), 1994, pp. 30238-30242
The primary specificity of papain-like proteinases is largely determin
ed by S-2-P-2 site interactions. According to the three dimensional st
ructure of a papain-inhibitor complex, the S-2 subsite is defined by r
esidues 67, 68, 133, 157, 160, and 205, with residues 133, 157, and 20
5 integrated into the wall and bottom of the side chain binding cavity
. The S-2 binding site specificity of this enzyme has been altered to
mimic that of cathepsin B or L by the application of site-directed mut
agenesis at these latter three positions in the cathepsin S sequence.
The replacement of Gly-133 in cathepsin S by an alanine residue that i
s normally found at this position in both cathepsin B and L results in
a pattern of specificity toward hydrophobic residues in P-2 that is v
ery similar to that of cathepsin B and L. The replacement of other cat
hepsin S S-2 subsite residues with their cathepsin L equivalents (muta
nts Val-157 --> Leu, Phe-205 --> Ala) does not significantly change th
e specificity of cathepsin S. Cathepsin B is distinguished hom both ca
thepsin L and S by its ability to efficiently hydrolyze substrates con
taining a basic P-2 residue. A single mutation in position 205 of cath
epsin S (Phe-205 --> Glu) results in a change of specificity toward th
at of cathepsin B, i.e. the second order rate constant for the hydroly
sis of the cathepsin B-specific substrate benzyloxycarbonyl-Arg-Arg-4-
methyl-7-coumarylamide is increased 77-fold for this mutant compared w
ith the wild-type enzyme. A cathepsin S double mutant Gly-133 --> Ala/
Phe-205 --> Glu is characterized by somewhat improved kinetic paramete
rs compared with the Phe-205 --> Glu single mutant. The hydrolysis rat
e of the benzyloxycarbonyl-Arg-Arg-4-methyl-7-coumarylamid substrate b
y this double mutant is 130-fold higher than that of the wildtype enzy
me. As with cathepsin B, the activities of the Phe-205 --> Glu single
and the Gly-133 --> Ala/Phe-205 --> Glu double mutants of cathepsin S
toward the dibasic substrate is modulated by an additional ionizable g
roup with a pK(alpha) of 5.7.