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  1. Prolidase deficiency (PD) (MIM 170100) is a rare, autosomal recessive, panethnic disorder associated with massive imidodipeptiduria. Undegraded dipeptides are excreted in excess of 15 mmol/day. Affected children often present with severe skin ulcers, particularly on their hands and feet. Mild to severe mental retardation occurs in approximately 75 percent of cases. Additionally, PD individuals appear to be highly susceptible to infections, some of which have been fatal. There is considerable clinical heterogeneity and at least five asymptomatic PD individuals have been identified. Three of these had severely affected sibs and two were detected in newborn screening programs.

  2. Prolidase (imidodipeptidase, peptidase D; EC is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of dipeptides with a C-terminal proline or hydroxyproline. The enzyme is encoded by the peptidase D (PEPD) gene located at 19q12-q13.11. PEPD spans 130 kb and has 15 exons. The 2.1 to 2.2 kb mRNA is translated into a 493-amino acid protein with a predicted molecular weight of 54.3 kDa. Native prolidase is a homodimer. A second, less well-characterized enzyme able to catalyze hydrolysis of imidodipeptides is known as prolidase II. Its activity does not appear to compensate for deficiency of prolidase.

  3. Prolidase is a metalloenzyme that is activated by Mn+2. The specificity and function of prolidase metal binding remains controversial. Recent reports indicate that Mn+2 incubation affects the V max but not the Km of prolidase and that Mn+2 also increases the thermostability of the enzyme. Some metals, such as Co+2, Mg+2 and Fe+2 stimulate enzyme activity to a lesser extent than Mn+2; others, such as Zn+2, Pb+2, Hg+2 and Cd+2, inhibit prolidase activity.

  4. A structural model for prolidase has been proposed based on homology modeling with crystallized E. coli methionine aminopeptidase. There is a “pita-bread fold” in which five amino acid residues form two metal binding sites in each subunit. This predicted stoichiometric ratio is supported by atomic absorption spectroscopy on purified, activated human prolidase. The catalytic site of prolidase is predicted to contain an arginine and an acidic amino acid.

  5. Mutation analysis in PD individuals has identified nine PEPD mutations. Most are CpG mutations and only one, G448R, has been found in multiple, unrelated individuals. Expression systems for testing the functional consequences of PEPD mutations use COS cells or NIH3T3 cells.

  6. Proline is a nonessential amino acid. Its biosynthesis in human tissues occurs via the action of Δ-1-pyrroline-5-carboxylic acid reductase upon glutamic semialdehyde (see Chap. 81). Both glutamic acid and arginine (via ornithine) are precursors. Studies in cultured fibroblasts have shown, however, that these sources contribute to less than 10 percent of collagen-bound proline. This suggests a role for prolidase in recycling dipeptide-bound proline back to the free amino acid pool for utilization in protein biosynthesis. Prolidase substrates can entirely satisfy the growth requirements of cultured CHO cells auxotrophic for proline.

  7. The abundance of glycyl-L-proline (typically 15 to 35 percent of excreted dipeptide) in the urine of PD patients and the presence of hydroxyproline-containing dipeptides suggests a role for prolidase in collagen turnover. The urine ofprolidase deficient patients contains high concentrations of glutamyl-L-proline and aspartyl-L-proline, dipeptides not abundant in collagen. This observation suggests that prolidase participates in the degradation of proteins other than collagen. Although prolidase is ubiquitous, both the intestine and the kidney have high prolidase activities. This suggests prolidase may be involved in the absorption of imidodipeptides. Consistent with this notion, studies in both humans and mice indicate a role for prolidase in dipeptide transport.

  8. Laboratory diagnosis of PD is straightforward. The amino acid profile of unhydrolyzed urine samples from prolidase-deficient individuals is normal. Acid hydrolyzed specimens, however, contain massive amounts of proline, hydroxyproline, and free amino acids derived from the N-terminal position of the dipeptide. Confirmation of diagnosis by assay of prolidase activity can be performed on erythrocytes, leukocytes, or cultured fibroblasts. Small and variable activity of a second enzyme, prolidase II, may obscure the difference between markedly decreased or absent prolidase activity.

  9. Current treatments for PD include dietary supplementation with L-proline; Mn+2 and ascorbic acid; supplementation with essential amino acids; erythrocyte transfusions; and topical application of ointments containing L-glycine and L-proline. Despite encouraging preliminary reports, none of these therapeutic approaches has achieved consistent success.

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