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 deficiency (McKusick 26413) is a rare autosomal recessive disorder characterized by iminodipeptiduria, skin lesions and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. / The results presented here indicate that prolidase plays a major role in the recycling of dipeptide bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth and protein synthesis whereas prolidase deficient cells were not. / Iminodipeptides added to the media of control and mutant cells showed no adverse effects on protein synthesis or cell growth. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is the cause of damage to skin cells. / Prolidase regulation by product and substrate was studied. A two fold decrease of prolidase activity was observed in fibroblasts grown in excess proline. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.

Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.

No description available.

Lactobacillus plantarum WCSF1 has two putative prolinases (PepR1 and PepR2), and they share only 48.5% amino acid sequence identity. To investigate the differences in enzymatic characters between two enzymes, the genes are cloned and expressed in E. coli using non-tagged pKK223-3 and His-tagged pET32b(+) systems. Culture conditions of overexpressed recombinant prolinases (r-PepR1 and r-PepR2) are optimized as pH7.0-7.5 LB media at 16°C with 1 mM IPTG induction. Recombinant prolinases with His-tag give higher yields and are more cost-efficient over non-tagged recombinant prolinases. After purification, these recombinant enzymes show similar hydrolysis activities towards Pro-Gly substrate, proving their nature as prolinases. Structural analyses using CD spectrum and computer modelling show that r-PepR1 and r-PepR2 share structural similarity in their secondary structure having the highest β-sheets over other components; and dynamic light scattering and gel filtration chromatography analyses indicate their quaternary structure being homotetrameric. Structural similarity can be linked to enzyme function feature. The two enzymes have the same enzymatic functionality may be due to their structural similarity. Despite for their structural similarities and the same enzymatic functionality, they show differences in their substrate specificity, optimum temperature and pH, kinetic parameters (Km and kcat values), thermal stability, and proteolysis mode. r-PepR1 has its optimal activity at 25°C pH7.5 against substrate Pro-Met, whereas r-PepR2 is most active at 30°C pH8.0 against Pro-Gly. r-PepR1 has a low thermal stability with a TM (the midpoint temperature of the unfolding transition) at 29°C, whereas r-PepR2 has a higher TM at 48°C. However, r-PepR1 is aggregated and inactivated at near physiological temperature (42°C). The catalytic mode of r-PepR1 could be a metallo-protease since its activity reduces by 38% with a metal-chelating agent EDTA; while the activity of r-PepR2 is inhibited by 47% with a serine protease inhibitor PMSF, suggesting it is a serine protease. These isozymes cooperatively and complementarily work together to hydrolyze proline-containing peptides, showing broader specificity, broader range of working pH and temperature, and higher efficiency, suggesting that the proline recycling are mediated through these two enzymes to adapt a wide rage of environmental conditions.