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Merck
  • 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.

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 Journal of biological chemistry (1993-01-05)
S Hasnain, T Hirama, C P Huber, P Mason, J S Mort
초록

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.

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Sigma-Aldrich
Cathepsin B Substrate, colorimetric