Renaturation of a multisubunit multiactivity enzyme complex: recovery of phage Qbeta RNA replicase, EF-Tu, and EF-Ts activities after denaturation in urea.
Phage Qbeta RNA replicase consists of four nonidentical subunits three of which are required for poly(C)-directed synthesis of poly(G): a phage-coded polypeptide and the two host-supplied protein biosynthesis elongation factors EF-Tu and EF-Ts. After denaturation of the enzyme in 8 M urea, poly(G) polymerase activity can be renaturated by dilution of the denatured subunits into a high ionic strength buffer with glycerol. The renaturation reaction has a broad temperature optimum between 11 and 21 degrees. The extent of renaturation is dependent on enzyme concentration: at low enzyme concentrations and 21 degrees renaturation proceeds for more than 3 h with greater than 40% recovery  of activity, whereas at high enzyme concentrations the reaction is complete by 1 h with less than 10% of the poly(G) polymerase activity regained. Activities catalyzed by the elongation factors can be measured while they are part of the replicase complex. Study of rates of renaturation of EF-Tu and EF-Ts dependent activities alone and in the replicase complex revealed that virtually 100% of the EF-Ts activity was recovered more rapidly than could be assayed at temperatures as low as 2 degrees, while the rate of recovery of EF-Tu activity was comparable to that of the poly(G) polymerase activity and was independent of either EF-Tu concentration or the presence of other enzyme subunits. The rate of recovery of the poly(G) polymerase activity was found to be limited by the renaturation of EF-Tu, since the rate was dramatically increased by the addition of undenatured EF-Tu.
D001665:Binding Sites; D003090:Coliphages / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D011485:Protein Binding; D011489:Protein Denaturation; D011777:Q beta Replicase / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology*
D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D014508:Urea; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases