Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

Three residues, Phe190, Leu193 and Val302, which have been proposed to define the S₁ site of prolidase of <i>Lactococcus lactis</i> NRRL B-1821 (<i>L. lactis</i> prolidase), may limit the size and polarity of specific substrates accepted by this enzyme (Yang, S. I., and Tanaka, T. 2008. Characterization of recombinant prolidase from <i>L. lactis</i> changes in substrate specificity by metal cations, and allosteric behavior of the peptidase. FEBS J. 275, 271-280). These residues form a hydrophobic pocket to determine the substrate specificity of <i>L. lactis</i> prolidase towards hydrophobic peptides, such as Leu-Pro and Phe-Pro, while little activity was observed for anionic Asp-Pro and Glu-Pro. It is hypothesized that the substrate specificity of <i>L. lactis</i> prolidase would be changed if these residues are substituted with hydrophilic amino acid residues individually or in combinations by site-directed mutagenesis (SDM). In addition to the changes in substrate specificity, other characteristics of wild type prolidase, such as allosteric behaviour and substrate inhibition may receive influences by the mutations (Yang & Tanaka, 2008). To test this hypothesis, mutations were conducted on these three residues at the S₁ site. Mutated <i>L. lactis</i> prolidases were subsequently analyzed in order to examine the roles of these residues in the substrate specificity, allosteric behaviour, pH dependency, thermal dependency and metal dependency of prolidase. The results showed the significant changes in these kinetic characteristics of single mutants, such as L193E, L193R, V302D and V302K and double mutants, L193E/V302D and L193R/V302D. Leu193 was suggested to be a key residue for substrate binding. The mutants L193R, V302D, L193R/V302D and L193E/V302D lost their allosteric behaviour, and the substrate inhibition of the wild type was no longer observed in V302D and L193E/V302D. The results indicated Val302 to be more important for these properties than other S₁ site residues. Moreover, together with the observations in molecular modelling of the mutants, it was proposed that interactions of Asp302 with Arg293 and His296 caused the loss of allosteric behaviour and substrate inhibition in the V302D mutant. The investigations on the pH dependency suggested that His296 acted as proton acceptor in <i>L. lactis</i> prolidase's catalysis. It was expected that the electrostatic microenvironment surrounding His296 was influenced by the charged mutated residues and side chains of dipeptide substrates, thus the protonation of His296 was affected. It was suggested that the introduced positive charge would stabilize the deprotonated form of His296 thus to maintain the activities of the mutants in more acidic condition compared to wild type prolidase. The study of thermal dependency revealed that all non-allosteric prolidases had higher optimum temperatures, suggesting that the loss of allosteric behaviour resulted in more rigid structures in these prolidases.

Prolidase has potential applications in cheese debittering, organophosphate detoxification and as an enzyme replacement therapy in prolidase-deficient patients. Recombinant Lactococcus lactis prolidases and their catalytic properties have previously been characterized in Dr. Tanaka's research group. Unlike other prolidases, L. lactis prolidase shows allosteric behaviour, metal-dependent substrate specificity and substrate inhibition. The current project focuses on elucidating the three-dimensional structure of L. lactis prolidase using X-ray crystallography. Hexagonal plate-like crystals of wild-type L. lactis prolidase were grown by the hanging drop vapour diffusion method, allowing the crystals to grow to about 50 µm in their longest dimension. The crystallization cocktail in which they grew contained 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, 14 % PEG 8000 and 18 % glycerol. Crystal diffraction data was collected at a wavelength of 0.9795 Å on beamline 08ID-1 of the Canadian Macromolecular Crystallography Facility at the Canadian Light Source and was processed using X-ray Detector Software. The crystals belonged to space group C2 and estimated to contain three molecules in an asymmetric unit. The electron density map of this structure was solved by the molecular replacement method and the structure model was refined against 2.25 Å resolution data. Molecule A forms a dimer with molecule B, while molecule C forms a dimer with molecule C', which is located in the neighbouring crystal asymmetric unit. The electron density of molecule A was well-defined and complete. Therefore, all the 362 amino acid residues of L. lactis prolidase were fitted. The other two molecules were incomplete and less defined. Only 360 and 352 residues could be fitted in molecules B and C, respectively. Molecule C, the worst of the three, compromised the overall quality of the refined structure. However, the functional interpretation of the structure was not compromised since the well-defined molecules form a dimer with each other and the biologically-functional form of L. lactis prolidase is a homodimer. The final Rwork and Rfree are 22.39 and 27.77, respectively. Comparison with other known prolidases revealed that Asp 36 and His 38 are unique to L. lactis prolidase. These residues have been shown to be involved in the allosteric behaviour and substrate inhibition of this enzyme, respectively. Therefore, this crystal structure further supports their suggested contribution in L. lactis prolidase's unique catalytic properties.