ENGINEERING THE S-2 SUBSITE SPECIFICITY OF HUMAN CATHEPSIN-S TO A CATHEPSIN-L-LIKE AND CATHEPSIN-B-LIKE SPECIFICITY

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.