Slow-Binding Inhibition of the Aminopeptidase from Aeromonas proteolytica by Peptide Thiols: Synthesis and Spectroscopic Characterization

Abstract

Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a-c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (K(I)) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a-b) are simple competitive inhibitors of much lower potencies (K(I) = 23 and 360 microM). These data suggest that the free thiols are involved in the formation of the E. I and E.I complexes, presumably serving as a metal ligand. To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c. Both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S --> Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified micro-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding. EPR spectra of [CoCo(AAP)]-1c, [ZnCo(AAP)]-1c, and [CoZn(AAP)]-1c were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW). The observed 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) approximately 6.8 that is characteristic of thiolate-Co(II) interactions. These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster. Compounds 1a-c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors.

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

“…The low temperature component was simulated as a single species with parameters consistent with a five-coordinate Co(II) center, although significant strain in E/D was required to obtain the unusual line shape. The persistence of this signal at 4K was suggestive, however, of an ${\mathrm{normalM}}_{\mathrm{normalS}}=\mid 1\frac{3}{2}\rangle$ signal (34, 35) and subtraction of the high temperature signal yielded a difference spectrum (Figure 5E) characterized by a spike at g eff. ∼6.3, consistent with ${\mathrm{normalM}}_{\mathrm{normalS}}=\mid 1\frac{3}{2}\rangle$ and a distorted tetrahedral geometry.…”

Identification of Human Glutaminyl Cyclase as a Metalloenzyme

“…Moreover, 1-butaneboronic acid and peptide thiols (22,23), potent inhibitors of V. proteolyticus aminopeptidase, did not inhibit human QC, supporting potential changes in the active site geometry of QC compared with the aminopeptidases. Because there have been extensive rearrangements during the evolution of the zinc hydrolase group, including remodeling of the active site upon changes in zinc ligation (20,21), only the solution of the protein structure will finally clarify the binding modes of substrate and inhibitor.…”

Bacterial protease inhibitors

“…156,157 Thus, inhibitors of such enzymes might be important for the design of new antibiotics. Indeed, Huntington et al 160 reported that peptide-derived thiols of the general structure 11 are potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica, with K I values in the range from 2.5 to 57 nM (Fig. 6).…”

“…6). To investigate the nature of the interaction of these thiol-based inhibitors with the dinuclear active site of AAP, the electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor have been recorded, 160 being shown that both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of such inhibitors, exhibited an absorption band centered at 320 nm characteristic of an S ! Co(II) ligand-metal charge-transfer band.…”

“…These data suggested that the thiolate moiety of the inhibitor may bind to either of the metal ions in the dinuclear active site of AAP, but does not bridge the dinuclear cluster. 160 These compounds constitute interesting lead molecules for developing more potent LAP inhibitors with potential antibiotic properties.…”