RESUMEN
A number of natural and synthetic flavonoids have been assessed previously with regard to their effects on the activity of cyclin-dependent kinases (Cdk1 and -2) related to the inhibition of cell cycle progression. On the other hand, the Cdk5/p35 system is of major importance in neuronal migration phenomena and brain development, and its deregulation is implicated in neurodegenerative diseases, particularly Alzheimer's. Here we show that some natural flavonoids inhibit the activity of the Cdk5/p35 system in the micromolar range, while others are practically inactive. Ring B-unsubstituted and highly methoxylated flavones were inactive or gave irreproducible results, and 6-methoxyapigenin and 6-methoxyluteolin were the most potent Cdk5 complex inhibitors within this series, while the common flavonols kaempferol and quercetin showed intermediate behavior. The reported crystal structure of the Cdk5 complex with its activator p25 was used for docking studies, which also led to the identification of the two 6-methoxyflavones, kaempferol and quercetin, as well as the untested 6-methoxy derivatives of kaempferol and quercetin and the corresponding 6-hydroxy analogues as compounds exhibiting a good fit to the active site of the enzyme.
Asunto(s)
Centaurea/química , Quinasas Ciclina-Dependientes/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Flavonoides/farmacología , Flavonoles/farmacología , Algoritmos , Quinasa 5 Dependiente de la Ciclina , Flavonoides/administración & dosificación , Flavonoides/química , Flavonoles/administración & dosificación , Flavonoles/química , Concentración 50 Inhibidora , Quempferoles/administración & dosificación , Quempferoles/química , Quempferoles/farmacología , Conformación Molecular , Estructura Molecular , Quercetina/administración & dosificación , Quercetina/química , Quercetina/farmacología , Relación Estructura-ActividadRESUMEN
The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by N(epsilon) of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213. We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys212 and Lys213, and for Mn2+ binding. deltaG and deltaH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu284 is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by
Asunto(s)
Ácido Glutámico/metabolismo , Lisina/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Secuencia Conservada , Ácido Glutámico/química , Ácido Glutámico/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Estructura Terciaria de Proteína , ProtonesRESUMEN
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.