RESUMEN
The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.
Asunto(s)
Bacillus/enzimología , Fosfatos/metabolismo , Ribonucleasas/biosíntesis , Secuencia de Aminoácidos , Bacillus/genética , Bacillus/fisiología , Proteínas Bacterianas , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Endorribonucleasas/genética , Genes Bacterianos , Datos de Secuencia Molecular , Ribonucleasas/genética , Esporas BacterianasRESUMEN
To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.