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Titolo:
Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor-enzyme complex
Autore:
Bienvenue, DL; Bennett, B; Holz, RC;
Indirizzi:
Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA Utah State UnivLogan UT USA 84322 pt Chem & Biochem, Logan, UT 84322 USA
Titolo Testata:
JOURNAL OF INORGANIC BIOCHEMISTRY
fascicolo: 1, volume: 78, anno: 2000,
pagine: 43 - 54
SICI:
0162-0134(20000115)78:1<43:IOTAFA>2.0.ZU;2-P
Fonte:
ISI
Lingua:
ENG
Soggetto:
ANGIOTENSIN-CONVERTING ENZYME; METHIONINE AMINOPEPTIDASE; PEPTIDE HYDROLYSIS; BETA-LACTAMASE; POTENT; SITES; MECHANISM; BESTATIN; DESIGN; METALLOENZYMES;
Keywords:
peptide hydrolysis; aminopeptidases; peptide-thiolates; kinetics; electronic absorption; electron paramagnetic resonance spectroscopy; cobalt; zinc;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
49
Recensione:
Indirizzi per estratti:
Indirizzo: Holz, RC Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA Utah State Univ Logan UT USA 84322 Biochem, Logan, UT 84322 USA
Citazione:
D.L. Bienvenue et al., "Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor-enzyme complex", J INORG BIO, 78(1), 2000, pp. 43-54

Abstract

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 alcohol L-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (K-1 = 17 mu M). These data suggest that the free thiol is likely involved in the formation of the E.I and E.I* complexes, presumably providing ametal ligand. In order to probe the nature of the interaction of LeuSH andLeuOH 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 metalions. 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 mu-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 alsorecorded at lower temperature (3.5-4.0 K) and high microwave power (50-553mW). These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19K, a very sharp feature at g(eff) similar to 6.5 that is characteristic ofthiolate-Co(LI) interactions. Combination of the electronic absorption andEPR 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 mu-S(R) bridge is established. (C) 2000 Elsevier Science Inc. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/11/20 alle ore 15:30:59