Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.

To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.

The cloned phoE genes from Enterobacter cloacae and Klebsiellu pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K . pneumoniue phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81 YO of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.The outer membrane of gram-negative bacteria functions as a molecular sieve. Pore-forming proteins in this membrane allow the passage of small, hydrophilic solutes up to a defined exclusion limit by a diffusion-like process [l, 21. In Escherichia coli K-12 the family of pore-forming proteins includes three members, the OmpF, OmpC and PhoE proteins. OmpF and OmpC proteins are synthesized constitutively under standard growth conditions. Their relative amounts are determined by the osmolarity of the growth medium [3]. The synthesis of PhoE protein is induced when cells are growing under phosphate limitation [4]. Both in vivo [5] and in vitro [6] experiments have shown that the pores formed by PhoE protein have a preference for anions, in contrast to the cation-selective OmpF and OmpC pores.Nucleotide sequence analysis of the ompF, ompC andphoE genes has revealed an extensive homology of approximately 60% in the primary structures of the three proteins. The porins are unusual among membrane proteins in that they lack hydrophobic sequences long enough to span the membrane. The functional unit is a trimer with a compact structure, lacking large extramembrane domains [lo]. It has been shown that in OmpF protein approximately two-thirds of the polypeptide chain is in the form of an antiparallel P-pleated sheet, with many 10 -12-residue-long membranespanning segments, which run approximately normal to the plane of the membrane [ll]. In view of the close homology between the OmpF, OmpC and PhoE proteins, a similar structure for all three pore proteins is to be expected. A pre- dominant p structure has also been found in two other outer membrane proteins, LamB and OmpA, which are unrelated to the porins [12]. We have studied the topology and structure-function relationships of PhoE protein by two different approac...