RESUMO
The hepatitis C viruses (HCVs) are a group of small enveloped RNA viruses that have been viewed as a leading cause of chronic hepatitis in humans. Infections by HCV represent a serious global health problem, because millions of people worldwide are infected and no efficient treatment is available at the present time. Since HCV was identified in 1989, considerable effort has been devoted to the discovery and development of novel molecules to treat HCV-related diseases. One of the approaches is the development of novel inhibitors that interrupt the normal functions of HCV NS5B, an RNA-dependent RNA polymerase essential to HCV replication. This review summarizes recent advances in the biochemical and structural understanding of HCV NS5B polymerase as well as in the development of antiviral agents targeting this important enzyme.
Assuntos
Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA Nucleotidiltransferases/metabolismoRESUMO
The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.
Assuntos
Superóxido Dismutase/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Humanos , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas Recombinantes , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , TitulometriaRESUMO
Glutamine 143 in human manganese superoxide dismutase (MnSOD) forms a hydrogen bond with the manganese-bound solvent molecule and is investigated by replacement using site-specific mutagenesis. Crystal structures showed that the replacement of Gln 143 with Ala made no significant change in the overall structure of the mutant enzyme. Two new water molecules in Q143A MnSOD were situated in positions nearly identical with the Oepsilon1 and Nepsilon2 of the replaced Gln 143 side chain and maintained a hydrogen-bonded network connecting the manganese-bound solvent molecule to other residues in the active site. However, their presence could not sustain the stability and activity of the enzyme; the main unfolding transition of Q143A was decreased 16 degrees C and its catalysis decreased 250-fold to k(cat)/K(m) = 3 x 10(6) M(-)(1) s(-)(1), as determined by stopped-flow spectrophotometry and pulse radiolysis. The mutant Q143A MnSOD and other mutants at position 143 showed very low levels of product inhibition and favored Mn(II)SOD in the resting state, whereas the wild type showed strong product inhibition and favored Mn(III)SOD. However, these differences did not affect the rate constant for dissociation of the product-inhibited complex in Q143A MnSOD which was determined from a characteristic absorbance at 420 nm and was comparable in magnitude ( approximately 100 s(-)(1)) to that of the wild-type enzyme. Hence, Gln 143, which is necessary for maximal activity in superoxide dismutation, appears to have no role in stabilization and dissociation of the product-inhibited complex.
