Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization.
The pH dependence of cathepsin B-catalyzed hydrolyzes is very complex. At least seven dissociable groups are involved in the binding and hydrolysis of 7-amido-4-methyl coumarin and p-nitroaniline (pNA)-based substrates containing a P1 Arg and either a Phe or Arg at the P2 position. By site-directed mutagenesis we show that a previous suggestion, that Arg202 is one of the groups which influences the pH dependence of cathepsin B-catalyzed hydrolysis of the Z-Arg-Arg-pNA substrate, is not valid. However, it was found that Glu245, which has a pKa of 5.1 in rat cathepsin B, is responsible for the S2-P2 specificity for Arg-containing substrates and controls the pH dependence of their hydrolysis. Furthermore, the data indicate that Glu245, which forms a hydrogen bond with the guanidinium group of the substrate's P2 Arg, contributes about 1.8 kcal/mol to transition state stabilization in the protonated state and about 0.6 kcal/mol in the deprotonated state. Mutation of Glu245 to Gln results in a 16-fold decrease in kcat but does not affect Km. While cathepsin B has a 7-fold preference for Phe over Arg at the P2 position of a substrate, binding of the aromatic side chain does not appear to be influenced by Glu245.
Hasnain
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Protein Structure and Design Section, National Research Council of Canada, Ottawa, Ontario.
Hirama
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Huber
C P
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Mason
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Mort
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D002401:Cathepsin B