Self-activation of guanosine triphosphatase activity by oligomerization ofthe bacterial cell division protein FtsZ

The essential bacterial cell division protein FtsZ (filamentation temperatu re-sensitive protein Z) is a distant homologue to the eukaryotic cytoskelet al protein tubulin, We have examined the GTP hydrolytic activity of Escheri chia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations gr eater than 1 mu M. At 5 mu M FtsZ, we have determined a K-m of 82 mu M GTP and a V-max of 490 nmol of P-i min(-1) (mg of protein)(-1). Hydrolysis of G TP requires Mg2+ and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation exp eriments show that FtsZ exists as 68% dimer and 13% trimer at 2 mu M total protein concentration. Chemical crosslinking of FtsZ also shows that monome r, dimer, trimer, and: tetramer species are present at higher (>2 mu M) Fts Z concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization, We also monitored the eff ect of nucleotide and metal ion on the secondary structure of FtsZ; nucleot ide yielded no evidence of structural changes in FtsZ, but both Mg2+ and Ca 2+ had significant effects on secondary structure. Taken together, our resu lts support the Hypothesis that Mg2+-dependent GTP hydrolysis by FtsZ requi res oligomerization of FtsZ, On the basis of these results and structural c omparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.