Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor-enzyme complex

The peptide inhibitor L-leucinethiol (LeuSH) was found to be a potent, slow -binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency (K-1*) of LeuSH was 7 nM while the corresponding alcoh ol L-leucinol (LeuOH) was a simple competitive inhibitor of much lower pote ncy (K-1 = 17 mu M). These data suggest that the free thiol is likely invol ved in the formation of the E.I and E.I* complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the elec tronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AA P)] in the presence of both inhibitors. In the presence of LeuSH, all three Co( II)-substituted AAP enzymes exhibited an absorption band centered at 2 95 nm, characteristic of a S --> Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded in the 450 to 700 nm region all sh owed changes characteristic of LeuSH and LeuOH interacting with both metal ions. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW ) indicated that, in a given enzyme molecule, LeuSH interacts weakly with o ne of the metal ions in the dinuclear site and that the crystallographicall y identified mu-OH(H) bridge, which has been shown to mediate electronic in teraction of the Co(II) ions, is likely broken upon binding LeuSH. EPR spec tra of [CoCo(AAP)]-LeuSH, [ZnCo(AAP)]-LeuSH, and [Co_(AAP)]LeuSH were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW). These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) similar to 6.5 that is characteristic of thiolate-Co(LI) interactions. Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both met al ions in the dinuclear active site of AAP. Since the spin-spin interactio n seen in resting [CoCo( AAP) ] is abolished upon the addition of LeuSH, it is unlikely that a mu-S(R) bridge is established. (C) 2000 Elsevier Scienc e Inc. All rights reserved.