RESUMEN
Human cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/Δflap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/Δflap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ß-hydroxyamino acid. hcSHMT/Δflap was less sensitive to THF inhibition than the WT (Ki of 0.65 and 0.27 mm THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/Δflap, indicating that the flap is important for THF binding. hcSHMT/Δflap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.
Asunto(s)
Glicina Hidroximetiltransferasa/química , Ligandos , Secuencias de Aminoácidos , Sitios de Unión , Estabilidad de Enzimas , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Mutagénesis , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato , Tetrahidrofolatos/química , Tetrahidrofolatos/metabolismoRESUMEN
BACKGROUND: Coumarin-6 is a lipophilic dye and is often used as a model in delivery system. OBJECTIVE: The aim of this study was to improve the nonstructured lipid carrier (NLC) system loading with lipophilic molecule, coumarin-6, and to investigate its characteristics in terms of physical stability and controlled release profile. MATERIALS AND METHODS: Initially, the selection of the coating polymer was observed. Then, the preparation of the conventional NLC-loaded coumarin-6 was compared to the modified NLC-loaded coumarin-6 via the probe sonication. The physical properties and stability were determined by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques. The release profile was established using fluorescent spectroscopic method. RESULTS AND DISCUSSION: The size and zeta potential measurement showed significant decrease in the size range of the modified NLC-loaded coumarin and the lower intensity of the surface charge compared to the NLC-loaded coumarin. The change of crystallinity observed from DSC and XRD techniques indicated the molecular dispersion of coumarin-6 in the lipid matrix of NLC. The FT-IR spectra were also proven that coumarin-6 was entrapped in the NLC molecule. The result showed comparable controlled release profile to the conventional preparation with no difference on the cytotoxicity level. CONCLUSIONS: The modified NLC delivery system, therefore, exhibited the acceptable potential as a nanocarrier.
