The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with proline residues at the C-terminus. The PepQ gene has been cloned, overexpressed and the enzyme purified to homogeneity. The kcat and kcat/Km values for the hydrolysis of Met-Pro are 109 s-1 and 8.4 x 105 M-1 s-1, respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group was assessed as substrates for this enzyme. The SP-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a kcat of 36 min-1 and a kcat/Km of 710 M-1 s-1. The corresponding RP-enantiomer was more slowly hydrolyzed with a kcat of 0.4 min-1 and a kcat/Km of 11 M-1 s-1. The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX. The pH-rate profiles for the wild-type E. coli prolidase using proline dipeptides as substrates were obtained. The roles of H346, H228, and E384 in the enzyme catalytic mechanism were also investigated by obtaining the pH-rate profiles for the mutants H346N, H228N, and E384Q. In an effort to clarify the mechanistic role of the interaction of the α-amino group of Xaa-Pro with metal at the enzyme active site, comparisons of the hydrolytic activity for Ala-Pro and 1-(1-oxopropyl)-L-proline, in which a hydrogen replaces the α-amino group of Ala-Pro, were performed.

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with proline residues at the C-terminus. The PepQ gene has been cloned, overexpressed and the enzyme purified to homogeneity. The kcat and kcat/Km values for the hydrolysis of Met-Pro are 109 s-1 and 8.4 x 105 M-1 s-1, respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group was assessed as substrates for this enzyme. The SP-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a kcat of 36 min-1 and a kcat/Km of 710 M-1 s-1. The corresponding RP-enantiomer was more slowly hydrolyzed with a kcat of 0.4 min-1 and a kcat/Km of 11 M-1 s-1. The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX. The pH-rate profiles for the wild-type E. coli prolidase using proline dipeptides as substrates were obtained. The roles of H346, H228, and E384 in the enzyme catalytic mechanism were also investigated by obtaining the pH-rate profiles for the mutants H346N, H228N, and E384Q. In an effort to clarify the mechanistic role of the interaction of the α-amino group of Xaa-Pro with metal at the enzyme active site, comparisons of the hydrolytic activity for Ala-Pro and 1-(1-oxopropyl)-L-proline, in which a hydrogen replaces the α-amino group of Ala-Pro, were performed.

Expression of human prolidase in E. coli: Kinetic and metal binding properties. Massive iminodipeptiduria, severe skin ulcers, and mild mental retardation mark Prolidase Deficiency, a rare autosomal recessive disorder. In vitro, Prolidase requires incubation with Mn 2+ for maximal activity, however, the in vivo role of metal in prolidase catalysis has not been clarified. / Human Prolidase cDNA was cloned into the bacterial expression vector pRSET C. The plasmid expresses a chimeric protein consisting of a hexahistidine tag and enterokinase site at the N-terminus of prolidase. This protein was purified to homogeneity. Several properties of the purified prolidase were examined. Prolidase preferentially bound Mn in vivo. In vitro incubation and atomic absorption studies revealed a positive correlation between activity and Mn2+ content, and a negative correlation between Zn content and activity. Prolidase has two distinct metal binding sites each binding two metals with different metal binding affinities towards Mn2+ and Zn2+. Prolidase thermostability was directly proportional to percent Mn2+ content. In addition, chelators inhibited prolidase activity.

No description available.

No description available.

Prolidase (E.C.3.4.13.9) hydrolyzes imidodipeptides. Prolidase deficiency (PD) (McKusick no. 170100) is an autosomal recessive disorder characterized by a highly variable clinical phenotype. Mutation analysis was performed on a panel of 10 PD cell lines. Single-stranded conformation polymorphism analysis (SSCP) analysis on four overlapping cDNA-PCR products covering the entire coding region of the prolidase gene revealed seven novel mutations: G $ to$ A,551 (R184Q); G $ to$ A,833 (G278D); G $ to$ A, 1342 (G448R); G $ to$ A, 1354 (E452K); delGAG, 1354-1356 (delE452); a deletion of exon 5; and a deletion of exon 7. We used inverse PCR to clone intronic regions flanking exons 5 and 7 and designed primers for conventional PCR of these regions of the genome in patients expressing mRNAs with deleted exons. Two splice acceptor site mutations were identified: a G $ to$ C, nt $-$1 of intron 4 and an A $ to$ G, nt $-$2 of intron 6. To assess the biochemical phenotypes of four of these mutations (R184Q, G278D, G448R, and delE452), we have designed a transient expression system for prolidase in COS-1 cells. The enzyme was expressed as a fusion protein carrying the HA1 epitope of influenza hemagglutinin, allowing its immunological discrimination from the endogenous enzyme. Expression of the R184Q mutation produced 7.4% of control enzymatic activity while the expression of the G278D, G448R and delE452 produced inactive enzymes. Western analysis of the R184Q, G278D and G448R prolidases revealed stable immunoreactive material whereas the delE452 prolidase was not detectable. Pulse-chase experiments revealed that the delE452 mutant protein was synthesized but unstable. / The R184Q allele is carried by an asymptomatic individual, suggesting that its residual activity may be sufficient to prevent the development of symptoms. The other alleles, G278D, G448R, and delE452 which completely abolish enzyme activity associate with the symptomatic form of the disorder. Interestingly, these substitutions are all located at or very close to the putative metal binding residues of prolidase. / We have cloned and sequenced the mouse prolidase cDNA. We have cloned a genomic DNA fragment which carries exons 2, 3, and 4 of the mouse prolidase gene. We constructed a vector for the targeted disruption of the prolidase gene in mouse embryonic stem cells, to create a mouse model for prolidase deficiency.

<i>Lactobacillus plantarum</i> (<i>Lb. plantarum</i>) NRRL B4496 and <i>Lactococcus lactis</i> (<i>Lc. lactis</i>) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant <i>Lb. plantarum prolidase</i>, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant <i>Lc. lactis prolidase</i>. <p>The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.

<i>Lactobacillus plantarum</i> (<i>Lb. plantarum</i>) NRRL B4496 and <i>Lactococcus lactis</i> (<i>Lc. lactis</i>) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant <i>Lb. plantarum prolidase</i>, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant <i>Lc. lactis prolidase</i>. <p>The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.

No description available.

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.