RESUMEN
A Cu,Co derivative of the Cu,ZnSOD from Photobacterium leiognathi, in which cobalt has been selectively substituted for zinc, has been prepared and spectroscopically investigated. The derivative shows three bands in the visible region at 530, 566, and 600 nm when copper is in the oxidized state. Reduction or depletion of the copper ion produce a shift of the band absorbing at 600 to 590 nm because of the detachment from copper of the imidazolate bridging the two metals when copper is in the oxidized state. Numerous isotropically shifted 1H NMR lines are observed when copper is oxidized, confirming the presence of the imidazolate bridge between the two metals. Comparison of the optical and the NMR spectra with those observed for the eukaryotic enzyme reveals the occurrence of slight but unambiguous differences diagnostic of a different degree of distortion of the metal cluster between the prokaryotic and eukaryotic enzymes.
Asunto(s)
Cobalto , Cobre , Photobacterium/enzimología , Superóxido Dismutasa/química , Conformación Molecular , Células Procariotas/enzimología , EspectrofotometríaRESUMEN
The catalytic activity of a mutant of Photobacterium leiognathi Cu, Zn superoxide dismutase in which the Glu59 residue, conserved in most bacterial variants of the enzyme, has been replaced by glutamine was investigated by pulse radiolysis. At neutral pH the enzyme was found to have a kcat/KM of 1.0 +/- 0.1 x 10(10) M-1s-1 the highest value ever found for any superoxide dismutase. Brownian dynamics simulation suggests that such a high value is due to an enhanced substrate attraction by the modified electric field distribution. The mutant is also characterized by an active-site widely accessible for the solvent, since iodide is able to interact with the copper atom with an affinity constant twice as high as that found in the native enzyme. The large solvent accessible surface of the copper site together with a favorable distribution of the protein-generated electric field gives rise to the most efficient enzyme ever found with activity close to the diffusion limit.
Asunto(s)
Ingeniería de Proteínas , Superóxido Dismutasa/metabolismo , Sustitución de Aminoácidos , Aniones/metabolismo , Sitios de Unión , Catálisis , Simulación por Computador , Cobre/metabolismo , Difusión , Concentración de Iones de Hidrógeno , Yoduros/metabolismo , Cinética , Concentración Osmolar , Photobacterium/enzimología , Photobacterium/genética , Protones , Radiólisis de Impulso , Solventes/metabolismo , Electricidad Estática , Superóxido Dismutasa/genética , Volumetría , Agua/metabolismoRESUMEN
Neutralisation by site-directed mutagenesis of four charged and highly conserved residues of the electrostatic loop of Cu,Zn superoxide dismutase from Xenopus laevis, involved in the electrostatic attraction of the substrate: Lys120-->Leu, Asp130-->Gln, Glu131-->Gln and Lys134-->Thr, gives rise to a mutant enzyme which displays an affinity for monovalent inhibitor anions, such as N3-, higher than that of the wild type. Analysis of 300 ps of molecular dynamics simulation carried out on the wild type and on the Xenopus laevis Cu,Zn superoxide dismutase mutant indicates that the two proteins display a distinct dynamical behaviour. In particular the root mean square deviation from the starting structure, the number of residues in random coil conformations, the number of residues in unfavourable regions of the Ramachandran plot indicate that the mutant displays a rigidity higher than the native enzyme. This is also evidenced by the loss of dynamical cross correlations in the simulation of the mutant, which on the other hand are present in the wild type. Moreover the mutant protein shows a different organisation of the backbone-to-backbone hydrogen bonds network that generates a rigid structure leading to an increase of the active site accessibility when compared to the native enzyme. It is suggested that the rigid state in which the mutant is confined, accompanied by the increase of the solvent accessible surface of the active site may explain the difference in reactivity toward the inhibitor anion.