Effects of Proline Analogues on the Formation of Alkaline Phosphatase in Escherichia coli

Abstract

Two of the four proline analogues tested for their effect on the formation and activity of Escherichia coli alkaline phosphatase were able to substitute for proline in protein synthesis in a proline auxotroph. One of these, 3, 4-dehydroproline, effectively replaced proline and led to formation of an active enzyme under conditions where no proline was present in the polypeptides. Substitution of azetidine-2-carboxylate for proline prevented active enzyme formation, producing instead altered monomeric forms of the alkaline phosphatase. These were detected with antibodies specific to denatured forms of the enzyme, and they were also characterized, together with cellular proteins, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Alkaline phosphatase, as well as several other proteins, is localized exterior to the bacterial cell cytoplasm in the periplasmic space. In the presence of azetidine-2-carboxylate, a substantial number of these periplasmic proteins retain their specific site of localization, and the denatured subunits of alkaline phosphatase were only detected in the periplasmic fraction of the cell. Thus, secretion of these proteins does not appear to require a high degree of specificity in the native structure of the polypeptide chain. The analogues 4-allohydroxyproline and 4-thiazolidine carboxylate were unable to substitute for proline in protein synthesis but they inhibited growth of E. coli.An important stage in the formation of the alkaline phosphatase of Escherichia coli (EC 3.1.3.1.) is the transport of the protein from its site of synthesis in the cytoplasm to its site of function in the periplasm, a region of the bacterial cell exterior to the cytoplasmic membrane. From studies with several alkaline phosphatase-negative mutants that synthesized antigenically related forms of the enzyme, we had determined that the protein was probably transported in the form of the subunit prior to dimerization and activation by metal (15). These latter two additional reactions are essential for production of an active enzyme. Support for this model of transport of the subunit came from observations that E. coli spheroplasts that were able to make protein but unable to form active alkaline phosphatase secreted subunits of this protein into the spheroplast medium (14). Mechanisms for the transport of large polypeptides across bacterial membranes have not been described, but any system of transport must possess a high degree of specificity because only a small frac-tion of bacterial proteins are localized outside the cytoplasm. We have attempted to determine whether the specificity for alkaline phosphatase transport lies in the secondary or tertiary structure of the polypeptide chain. Because protein structure depends upon the specific amino acid sequence in the polypeptide, we altered the primary structure with analogues of amino acids that were able to substitute for the naturally occurring amino acid in protein synthesis. Previous experiments with two histidine analogues (1,2, 4-triazole-3-alanine and ...

Pleiotropic Effects of Mutations Involved in the Regulation of Escherichia coli K-12 Alkaline Phosphatase

“…These data help to explain our previous results describing increases in the amount of proteins in K-12 periplasmic fractions after starvation cultures for P1 (15). During exponential growth, about 5 to 7% of the cell protein can be found in the periplasmic fraction but this value increases to 15 to 20% upon starvation for P. Only one-third of the increase could be attributed to the alkaline phosphatase.…”

Roles of extensins in cotyledon primordium formation and shoot apical meristem activity in Nicotiana tabacum

“…Unlike most other proline analogues, the potent inhibitor 3,4-DHP functions at micromole concentrations to inactivate prolyl hydroxylase in vivo rapidly and irreversibly (Cooper and Vamer, 1983). Studies with an Escherichia coli proline auxotroph demonstrated that 3,4-DHP was the only Pro analogue tested that could replace proline in the synthesis of enzymically active acid phosphatase ( Morris and Schlesinger, 1972 ). For 3,4-DHP offers many advantages over other inhibitors, so it becomes a useful tool for studying the physiological and developmental functions of extensins among all types of HRGPs.…”

Localization of Polyribosomes Containing Alkaline Phosphatase Nascent Polypeptides on Membranes of Escherichia coli

“…Bacteria also secrete proteins from the cell cytoplasm; in some strains these proteins appear in the culture medium whereas in others the polypeptides are retained in a region of the cell envelope referred to as the periplasmic space (23). In exponentially growing cultures of Escherichia coli K-12, 5 to 8% of the cell protein is in the periplasm (15,22). This value increases twoto threefold in E. coli mutants that have become constitutive for production of alkaline phosphatase, a normally repressed, periplasmic enzyme.…”

“…This value increases twoto threefold in E. coli mutants that have become constitutive for production of alkaline phosphatase, a normally repressed, periplasmic enzyme. About half of the increase can be accounted for by the alkaline phosphatase alone (22).…”