Asunto(s)
Fragmentos de Péptidos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Anhidrasas Carbónicas/química , Cromatografía de Afinidad , Clonación Molecular , Enteropeptidasa/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/químicaRESUMEN
Peptide analogs which correspond to the conserved region of the natural ATPase inhibitor protein from beef heart, Candida utilis, and Saccharomyces cerevisiae mitochondria were synthesized by solid-phase methodologies and tested for ATPase inhibitory activity. These peptides were found to be potent inhibitors of F1-ATPase-catalyzed ATP hydrolysis in acidic reaction media, having I50 values of 1.1 +/- 0.4 microM, 10 +/- 5 microM, and 48 +/- 19 microM, respectively. These results closely match those obtained for the naturally occurring inhibitor proteins. Additional peptides that correspond to the beef heart beta-subunit near the binding site of the beef heart inhibitor protein and that possess a substantial homology with the conserved region of the inhibitor protein were synthesized. Several of these peptides were found to be inhibitors of the ATPase activity. The best inhibitor, with an I50 value of 20 +/- 3 microM, was the peptide resembling the beef heart beta-subunit comprising amino acids 394-413. This peptide most closely resembles the peptides derived from the conserved region of the inhibitor protein. The insertion of five glycine residues between the charge clusters in the beta-394-413 peptide resulted in a peptide which was able to stimulate the hydrolysis of ATP.
Asunto(s)
Mitocondrias Cardíacas/química , Fragmentos de Péptidos/síntesis química , Proteínas/química , ATPasas de Translocación de Protón/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Candida/química , Bovinos , Concentración de Iones de Hidrógeno , Cinética , Mitocondrias/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/química , Proteína Inhibidora ATPasaRESUMEN
The cholesterol esterase and lipoprotein lipase catalyzed hydrolyses of the water-soluble substrate p-nitrophenyl butyrate are competitively inhibited by butaneboronic acid and phenylboronic acid. Phenyl-n-butylborinic acid has been synthesized and characterized as an ultrapotent transition state analog inhibitor: Ki = 2.9 +/- 0.6 nM and 1.7 +/- 0.3 microM for the cholesterol esterase and lipoprotein lipase reactions, respectively. These results are interpreted in terms of transition state structure and stabilization.
Asunto(s)
Ácidos Borínicos/farmacología , Compuestos de Boro , Lipólisis/efectos de los fármacos , Animales , Ácidos Borónicos/farmacología , Butiratos/farmacología , Cinética , Lipoproteína Lipasa/metabolismo , Matemática , Esterol Esterasa/metabolismo , PorcinosRESUMEN
The mechanism of cholesterol esterase- (carboxylic ester hydrolase, EC 8.1.1.1) catalyzed hydrolysis of the water-soluble ester p-nitrophenyl butyrate has been characterized for commercially available preparations from bovine and porcine pancreas and for a purified preparation from porcine pancreas. Kinetic evidence for an acylenzyme mechanism is provided by experiments wherein the butyryl enzyme is trapped by MeOH, EtOH or n-BuOH. For the last alcohol the transacylation product n-butyl n-butyrate was characterized by GC-mass spectrometry. Solvent isotope effects have been measured for Vmax/Km, which is the rate constant for acylation, and for Vmax, which monitors rate-determining deacylation. Isotope effects of 1.5-3 on these rate constants indicate that both steps of the acylenzyme mechanism for cholesterol esterase catalysis involve transition states that are stabilized by general acid-base proton bridges.