RESUMEN
The catalytic strategies of enzymes (such as citrate synthase) whose reactions require the abstraction of the alpha-proton of a carbon acid remain elusive. Citrate synthase readily catalyzes solvent proton exchange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloacetate. Because no further reaction occurs with this analog, it provides a uniquely simple probe of the roles of active site interactions on carbon acid proton transfer catalysis. In view of the high reactivity of the analog for proton transfer to the active site base, its failure to further condense with oxaloacetate to form a sulfur-less analog of citryl-coenzyme A was unexpected, although we offer several possible explanations. We have measured the rate constants for exchange, k(exch), at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues. Comparisons between the values of k(exch) are straightforward in two limits. If the rate of exchange of the transferred proton with solvent protons is rapid, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate constants for proton transfer. If the exchange of the transferred proton with protons in the bulk solution is the slow step and the equilibrium constant for proton transfer is unfavorable (as is likely), then k(exch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bulk solvent. If that exchange rate with bulk solution remains constant for a series of mutant enzymes, then k(exch) values compare the equilibrium constants for proton transfer. The importance of the acetyl-CoA site residues, H274 and D375, is confirmed with D375 again implicated as the active site base. The results with the series of oxaloacetate site mutants, H320X, strongly suggest that activation of the first substrate, oxaloacetate, through carbonyl bond polarization, not just oxaloacetate binding in the active site, is required for the enzyme to efficiently catalyze proton transfer from the methyl group of the second substrate.
Asunto(s)
Acetilcoenzima A/metabolismo , Citrato (si)-Sintasa/metabolismo , Acetilcoenzima A/química , Animales , Sitios de Unión , Dicroismo Circular , Citrato (si)-Sintasa/genética , Clonación Molecular , Cartilla de ADN/química , Escherichia coli/genética , Expresión Génica/genética , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Mutación , Miocardio/enzimología , Oxaloacetatos/metabolismo , Protones , PorcinosRESUMEN
Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.
Asunto(s)
Actinas/química , Proteínas Fúngicas/química , Gelsolina/química , Concentración de Iones de Hidrógeno , Cinética , Mutación Puntual , Polímeros , Unión Proteica , Saccharomyces cerevisiae/química , Relación Estructura-Actividad , TemperaturaRESUMEN
The genome of Sindbis virus encodes the polypeptides that are required for the replication and transcription of the virus RNA in infected cells. These polypeptides are translated as a polyprotein that is co- and post-translationally cleaved by an autoproteinase to give rise to four polypeptides designated nsP1, nsP2, nsP3 and nsP4. We have initiated a study of the functions of these proteins by expressing them in the Autographa californica baculovirus polyhedrin expression system. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized the four Sindbis polypeptides. We used a complementation assay which measures chloramphenicol acetyltransferase (CAT) activity to demonstrate that these proteins were biologically active. The infected cells were transfected with a Sindbis defective RNA that contains the CAT gene downstream of the promoter for the synthesis of the viral subgenomic RNA. CAT activity was found only in cells that had been infected with the recombinant baculovirus, not with wild type baculovirus, indicating that the required Sindbis nsP activities were present. Sindbis virions grew poorly in S. frugiperda cells and self-replicating Sindbis RNAs produced only very low levels of biological activity. Our results suggest that these cells are defective in their ability to replicate Sindbis RNAs and that the block is partially overcome when the Sindbis nsP mRNA is expressed under the control of the baculovirus DNA.
Asunto(s)
Baculoviridae/genética , Cápside/genética , Vectores Genéticos/genética , Virus Sindbis/genética , Proteínas del Núcleo Viral/genética , Animales , Secuencia de Bases , Cápside/biosíntesis , Células Cultivadas , Expresión Génica , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Infecciones por Togaviridae/genética , Proteínas del Núcleo Viral/biosíntesis , Proteínas no Estructurales ViralesRESUMEN
Endonuclease activity identified in crude preparations of rat and human heart mitochondria has each been partially purified and characterized. Both the rat and human activities purify as a single enzyme that closely resembles the endonuclease of bovine-heart mitochondria (Cummings, O.W. et. al. (1987) J. Biol. Chem. 262:2005-2015). All three enzymes, for example elute similarly during gel filtration and DNA-cellulose chromatography, and exhibit similar enzymatic properties. Although the nucleotide sequences of the mtDNAs indicate that there has occurred an unusual degree of divergence in the displacement-loop region during mammalian evolution, the nucleotide specificities of the mt endonucleases appear highly conserved and show a striking preference for an evolutionarily-conserved sequence tract that is located upstream from the heavy (H)-strand origin of DNA replication (OriH).
Asunto(s)
ADN Mitocondrial/metabolismo , Endodesoxirribonucleasas/metabolismo , Mitocondrias Cardíacas/enzimología , Animales , Secuencia de Bases , Evolución Biológica , Cromatografía de Afinidad , Cromatografía en Gel , ADN de Cadena Simple/metabolismo , Genes , Humanos , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The RNA polymerase of HeLa cell mitochondria has been purified free of endonuclease and DNA topoisomerase activities, permitting evaluation of the effect of template topology on transcription in vitro. On single-stranded DNA templates, transcription is nonspecific and does not require mitochondrial DNA sequences. In contrast, duplex DNA templates are efficiently transcribed only when they (1) carry the mitochondrial D-loop region and (2) are negatively supercoiled. These findings suggest a role for template superhelicity in modulating mitochondrial transcription in vivo.