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

• Published in: Biochemistry (Easton) Vol. 38; no. 47; pp. 15587 - 15596
• Main Authors: Huntington, Kristi M, Bienvenue, David L, Wei, Yaoming, Bennett, Brian, Holz, Richard C, Pei, Dehua
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23-11-1999
• Subjects: Aeromonas - enzymology; Aeromonas proteolytica; aminopeptidase; Aminopeptidases - antagonists & inhibitors; Aminopeptidases - chemistry; Bacterial Proteins; Binding, Competitive; Cobalt - chemistry; Dipeptides - chemical synthesis; Dipeptides - pharmacology; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - pharmacology; Kinetics; Leucine - chemistry; Leucine - pharmacology; Metalloproteins - chemistry; N-Mercaptoacyl-leucyl-@ip-nitroanilide; peptide thiols; Peptides - chemical synthesis; Peptides - pharmacology; Spectrophotometry; Sulfhydryl Compounds - chemical synthesis; Sulfhydryl Compounds - pharmacology; thiols; Zinc - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi991283e

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 μM). 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 μ-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 ≈ 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.