No abstract

Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

Beta-APP cleaving enzyme (BACE) is responsible for the first of two proteolytic cleavages of the APP protein that together lead to the generation of the Alzheimer's disease-associated Abeta peptide. It is widely believed that halting the production of Abeta peptide, by inhibition of BACE, is an attractive therapeutic modality for the treatment of Alzheimer's disease. BACE is an aspartyl protease, and there is significant effort in the pharmaceutical community to apply traditional design methods to the development of active site-directed inhibitors of this enzyme. We report here the discovery of a ligand binding pocket within the catalytic domain of BACE that is distinct from the enzymatic active site (i.e., an exosite). Peptides, initially identified from combinatorial phage peptide libraries, contain the sequence YPYF(I/L)P(L/I) and bind specifically to this exosite, even in the presence of saturating concentrations of active site-directed inhibitors. Binding of peptides to the BACE exosite leads to a concentration-dependent inhibition of proteolysis for APP-related, protein-based substrates of BACE. The discovery of this exosite opens new opportunities for the identification and development of novel and potentially selective small molecule inhibitors of BACE that act through exosite, rather than active site, binding interactions.

The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.