Studies on the Aminopeptidase Activity of Rat Cathepsin H

Three synthetic substrates H‐Arg‐NH‐Mec, Bz‐Arg‐NH‐Mec and H‐Cit‐NH‐Mec (Bz, benzoyl; NH‐Mec, 4‐methylcoumaryl‐7‐amide; Cit, citrulline) were used to characterize specificity requirements for the P 1 ‐S 1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that K m , k cat and the specificity constants k cat / K m are quite similar for substrates with a free α‐amino group. In contrast, a 25‐fold decrease of k cat / K m was observed for the N‐terminal‐blocked substrate Bz‐Arg‐NH‐Mec. The activation energies for H‐Arg‐NH‐Mec and Bz‐Arg‐NH‐Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy ΔΔG b of the charged α‐amino group was estimated to ‐8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H‐Arg‐NH‐Mec and H‐Cit‐NH‐Mec was further investigated by inspection of the pH dependence of k cat / K m . The curves of the two substrates with a charged α‐amino group showed identical bell‐shaped profiles which both exhibit p K a1 and p K a2 values of 5.5 and 7.4, respectively, at 30°C. The residue with a p K a1 of 5.5 in the acid limb of the activity profile of H‐Arg‐NH‐Mec was identified by its ionization enthalpy Δ H ion = 21 kJ/mol as a β‐carboxylate or γ‐carboxylate of the enzyme, whereas the residue with a p K a2 of 7.4 was assigned to the free α‐amino group of the substrate with a Δ H ion of 59 kJ/mol. Bz‐Arg‐NH‐Mec showed a different pH‐activity profile with a p K a1 of 5.4 and a p K a2 of 6.6 at 30°C. Cathepsin H exhibits no preference for a basic P 1 side chain as has been shown by the similar kinetics of H‐Arg‐NH‐Mec and the uncharged, isosteric substrate H‐Cit‐NH‐Mec. In summary, specific interactions of an aniouic cathepsin H active site residue with the charged α‐amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidasc.