Modification of S1 subsite specificity in the cysteine protease cathepsin B.
Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases.
Fox
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Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Mason
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P
Storer
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AC
Mort
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JS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055316:Cystatin C; D015891:Cystatins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity
C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C050874:glycyl endopeptidase; D007930:Leucine; C024974:E 64