Hasnain S, Hirama T, Huber C P, Mason P, Mort J S
Protein Structure and Design Section, National Research Council of Canada, Ottawa, Ontario.
J Biol Chem. 1993 Jan 5;268(1):235-40.
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.
组织蛋白酶B催化水解的pH依赖性非常复杂。至少有七个可解离基团参与了7-氨基-4-甲基香豆素以及基于对硝基苯胺（pNA）且在P1位为精氨酸、P2位为苯丙氨酸或精氨酸的底物的结合与水解过程。通过定点诱变，我们发现之前的一种观点，即Arg202是影响组织蛋白酶B催化Z-Arg-Arg-pNA底物水解的pH依赖性的基团之一，是不正确的。然而，研究发现，在大鼠组织蛋白酶B中pKa为5.1的Glu245，决定了对含精氨酸底物的S2-P2特异性，并控制其水解的pH依赖性。此外，数据表明，与底物P2位精氨酸的胍基形成氢键的Glu245在质子化状态下对过渡态稳定的贡献约为1.8千卡/摩尔，在去质子化状态下约为0.6千卡/摩尔。将Glu245突变为Gln会导致kcat降低16倍，但不影响Km。虽然组织蛋白酶B对底物P2位的苯丙氨酸与精氨酸的偏好性为7倍，但芳香侧链的结合似乎不受Glu245的影响。
