| Despite the importance of processive enzymes, determinants of processivity are poorly understood. This thesis characterizes polyphosphate binding by processive E. coli exopolyphosphatase (PPX), investigating the number and location of binding sites, the role of these sites in processivity and the physical nature of binding.;The number and location of binding sites was probed with recovery of homogenous, moderate-chain-length intermediates at high salt concentration. These polyphosphate intermediates, 50-, 14-, and 2-phosphates in length, implied multiple binding sites on PPX extending roughly 100 A from the active site.;The role and location of the binding sites was investigated further with limited proteolysis of PPX. Two stable domains were found, with the N-terminal domain corresponding closely to the PPX region homologous to the sugar kinase/actin/hsp70 superfamily. When generated with PCR and overexpressed separately, these domains lacked polyphosphatase activity. However, a nonprocessive N-terminal active site was confirmed using nested C-terminal deletions of PPX. These deletions also indicated the N-terminus of PPX contained the binding sites responsible for the 14- and 2-residue polyphosphates, while the 50-residue site was on the C-terminus.;An assay was developed to quantify polyphosphate binding by PPX and its purified N- and C-terminal domains. This revealed PPX-polyphosphate binding to be due largely to the C-terminal domain of PPX as polyphosphate binding by purified C-terminal domain and native PPX were nearly identical.;The nature of the PPX-polyphosphate interaction was investigated through the effects of salt concentration on polyphosphate binding by PPX. In the range of 150 to 350 mN, the concentration of many types of salts had no effect on PPX-polyphosphate binding. Salt concentration effects, however, were dependent on the nature of the salt and in particular the nature of the anion. Both the anion dependence and the weak salt-concentration effects differ from the characteristics of nearly all nonspecific, coulombic interactions of proteins with polynucleotides. This is despite the similarity of polyphosphate to the phosphate backbone of polynucleotides and indicates PPX-polyphosphate binding may involve a novel mechanism. The results for PPX-polyphosphate binding are similar only to the helix-destabilizing proteins SSB of E. coli and gp32 of bacteriophage T4. |
