Inhibition of the Aminopeptidase from Aeromonas proteolytica by l-leucinethiol: Kinetic and Spectroscopic Characterization of a Slow, Tight-binding Inhibitor–enzyme Complex

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12 p.

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Journal of Inorganic Biochemistry

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doi: 10.1016/S0162-0134(99)00203-2


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 (KI*) of LeuSH was 7 nM while the corresponding alcohol l-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (KI=17 μM). These data suggest that the free thiol is likely involved 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 electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] in the presence of both inhibitors. In the presence of LeuSH, all three Co(II)-substituted AAP enzymes exhibited an absorption band centered at 295 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 showed 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 one of the metal ions in the dinuclear site and that the crystallographically identified μ-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon binding LeuSH. EPR spectra 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 geff∼6.5 that is characteristic of thiolate-Co(II) interactions. Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both metal ions in the dinuclear active site of AAP. Since the spin–spin interaction seen in resting [CoCo(AAP)] is abolished upon the addition of LeuSH, it is unlikely that a μ-S(R) bridge is established.


Journal of Inorganic Biochemistry, Vol. 78, No. 1 (January 15, 2000): 43-54. DOI.

Brian Bennett and Richard Holz were affiliated with Utah State University at the time of publication.