RESUMEN
The effect of various covalent chemical modifications on the transesterification activity and stability of adsorbed lipase B from Candida antarctica (CALB) was studied in 2-butanone and o-xylene. CALB species modified with either polyethylene glycol 2000 monomethyl ether (MPEG), polyethylene glycol 300 mono-octyl ether (OPEG) or n-octanol (OCT) were used in combination with a hydrophobic (Accurel) and a hydrophilic (Duolite) support. The thermostabilities of adsorbed CALB in both solvents, and that of free CALB in o-xylene were not influenced by the modifications. In contrast, the thermostability of free CALB in 2-butanone decreased 2.5-fold after MPEG modification and increased 1.5-fold after modification with OPEG and n-octanol, compared to that of native CALB. The activities of the native and modified CALB species were up to 9-fold higher after adsorption onto Accurel than those of the corresponding free enzymes. Adsorption of these enzyme species onto Duolite only resulted in a 2- to 3-fold increase in the activity of OPEG- and OCT-modified CALB. The modified CALB species adsorbed onto Accurel show similar or up to 2-fold lower activities than do native adsorbed CALB species, while 1.5- to 6-fold higher activities were found for modified CALB species adsorbed onto Duolite. We propose that hydrophobic modifiers induce conformational changes of CALB during adsorption on a hydrophobic support whereas all three modifiers protect CALB from structural alterations during adsorption onto a hydrophilic support.
Asunto(s)
Candida/enzimología , Enzimas Inmovilizadas/efectos de los fármacos , Enzimas Inmovilizadas/metabolismo , Lipasa/efectos de los fármacos , Lipasa/metabolismo , Absorción , Resinas de Intercambio de Catión/metabolismo , Dextranos/farmacología , Proteínas Fúngicas/efectos de los fármacos , Proteínas Fúngicas/metabolismo , Octanoles/farmacología , Polietilenglicoles/metabolismo , Polietilenglicoles/farmacología , Pliegue de Proteína , Temperatura , Factores de TiempoRESUMEN
The present study was undertaken to examine the relationship between chemical structure (steric and electrostatic fields) and affinity for the small intestinal oligopeptide carrier (PepT1) using comparative molecular field analysis (CoMFA), a three-dimensional approach towards building quantitative structure-activity relationships. Various biological activity parameters (Kt, Jmax, Pc) and molecular descriptors (CoMFA fields, isobutylalcohol/water distribution coefficients) were examined. The resulting field map provides information on the geometry of the binding site cavity and the relative weights of various properties in different site pockets for each of the substrates considered. The results indicate that carrier permeability (Pc), calculated as the ratio of the half-maximal concentration (Kt) and the maximal carrier flux (Jmax), is sensitive to composition, size and hydrophobicity of the ligands. The best model obtained showed a high correlation between the carrier permeability (Pc) and the steric (76.3% contribution) and electrostatic (23.7% contribution) molecular fields with a cross-validated r2 (q2) of 0.754. The model fitted the experimental data with a correlation coefficient of 0.993 and a standard error of 0.041, while the regression line between experimental and calculated Pc had a slope of 0.994 with an intercept of 0.009. These results lead to a better understanding of the molecular requirements for optimal drug-carrier interactions with the intestinal peptide transporter and offers a useful visual aid for designing new potentially interesting structures with affinity for the oligopeptide transporter PepT1.
Asunto(s)
Proteínas Portadoras/química , Intestino Delgado/metabolismo , Simportadores , Sitios de Unión , Transporte Biológico , Permeabilidad de la Membrana Celular , Ligandos , Modelos Moleculares , Estructura Molecular , Oligopéptidos/metabolismo , Transportador de Péptidos 1 , Electricidad Estática , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.