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A catalytic triad of Escherichia coli outer membrane phospholipase A (OMPLA) has been studied using site-directed mutagenesis for information about the importance of the catalytic Asn156. It is proposed that the coordination by the cofactor calcium of the putative oxyanion through two water molecules may decrease the importance of the asparagine in the catalytic triad.

Research has been conducted on the pancreatic phospholipase A. The structural basis for the K (super)* (sub)cat activation by the anionic interface is discussed.

Modification experiments of the substrate specificity of lipases from both Staphylococcus hyicus (SHL) and S. aureus (SAL) have been conducted. The experiments resulted in the generation of six point mutants, including G293N, T297P, E295F, L300I, K298F and I299V. Substitution of the F298 mutant by a lysine in SAL, which is hardly active on phospholipid substrates, resulted in a four-fold increase from previous phospholipase activity.

A study on gram-negative bacterial outer membrane phospholipase A (OMPLA) activity investigated whether or not a dimerization process is responsible for triggering OMPLA activity in membranes using fluorescence resonance energy transfer. Results of the study were analyzed within the context of the regulation of OMPLA activity by the surrounding lipid phase. Experimental methodology, results and conclusions are presented.

A series of alpha-keto triglyceride analogues that inhibit the lipase of Staphylococcus hyicus has been synthesized. Nonhydrolyzable ether, carbamoyl or amide bonds were used in replacing ester bonds to forestall hydrolysis at positions 1 and 2. An alpha-keto fatty acid was also introduced at position 3 which may have inhibited the lipase. It was observed that an ester or a hydroxyl on position 3 had no effect on the lipase. The analogues react with the active site Ser of the lipase and copy the tetrahedral intermediate involved in substrate hydrolysis.

Research was conducted to find out the effect of the presence of high-affinity binding site for calcium in Staphylococcus hyicus. Findings show that calcium binding does indeed stabilize the structure of staphylococcus lipases and even other types of lipases. Furthermore, the production of variants that are calcium-independent is possible with constrained structural homology with some other lipase as the basis.

A study was conducted to analyze the substrates of Staphylococcus aureus lipase and Staphylococcus hyicus lipase which were found to be homologous enzymes. The biochemical characteristics of these lipases were examined through in vivo chimeragenesis. Results revealed that Staphylococcus hyicus lipase exhibited a high phospholipase activity, hydrolyses neutral lipids while Staphylococcus aureus lipase degraded short-chain fatty acid esters.

Several models have been developed to explain the interfacial activation of lipolytic enzymes, grouped into substrate and enzyme models. P-nitrophenyl N-alkylcarbamates were synthesized with different alkyl chains to study the differences in chain length selectivity between Staphylococcus hyicus (SHL) and S. aureus (SAL). The results show that carbamates are useful in the investigation of the influence of calcium ions on carbamylation and decarbamylation.

The epitope insertion technique was employed to determine the topology of the outer membrane phospholipase A (OMPLA) of Salmonella typhimurium. Missense mutations were inserted at different locations in the pldA gene, encoding OMPLA, to form unique BamHI sites. These sites were then utilized to introduce linkers, encoding a 16-amino-acid B-cell epitope. The accessibility of inserted epitopes was determined. Three of seven predicted loops were found to be cell surface exposed.

The catalysis function of the two serine residues, Ser144 and Ser152 in the outer membrane phospholipase A (OMPLA) of Escherichia coli, was studied by site-directed mutagenesis. Substitution of cysteine for Ser144 caused one percent residual activity but the other replacements virtually produced inactive enzymes. Replacement of Ser152 by threonine or asparagine caused 40% and 2% residual activity, respectively, but other substitutions reduced enzymatic activity.

An analysis of the enterobacterial pldA gene of Escherichia coli which encodes the outer membrane phospholipase A involved the cloning and sequencing of pldA genes of Proteus vulgaris, Salmonella typhimurium and Klebsiella pneumoniae. Expression of cloned genes in E. coli and enzyme activity of the gene products was evident.

A study was conducted on the characterization of calcium binding to outer membrane phospholipase A (OMPLA), focusing on the role of Ca in the activation and dimerization processes of OMPLA. Results suggested the presence of two Ca binding sites per OMPLA molecule. These sites may contain at least one negatively charged carboxylate. The primary structure of OMPLA had 15 glutamic acid and 16 aspartic acid residues. These residues were found to be the most likely candidates for being calcium ligands.

Research was conducted to examine the Fusarium solani cutinase in complex with phosphonate inhibitors using several nuclear magnetic resonance approaches to investigate internal motions on various time scales. A comparison of results with those of dynamical studies was carried out on free cutinase. Findings indicate that cutinase does need conformational rearrangements to bind its substrate which may develop the rate-limiting step in catalysis.