Recombinant Purine nucleoside phosphorylase (punA)
Description
Definition and Biological Role
Recombinant PNP is a salvage pathway enzyme that catalyzes the reversible reaction:
It is essential for nucleotide recycling, particularly in tissues lacking de novo purine synthesis, such as the brain . Deficiencies in PNP cause severe T-cell immunodeficiency and neurological disorders due to toxic dGTP accumulation .
Key Kinetic Parameters
| Substrate | Specific Activity (U/mg) | Source |
|---|---|---|
| Inosine | ≥25 | Human, E. coli |
| 7-Methyl-6-thioguanosine | 0.2–0.3 | E. coli |
| 2'-Deoxyfluoro nucleosides | 0.01–0.03 | Thermophiles |
Assay Protocol:
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Substrate: 400 µM 7-methyl-6-thioguanosine (MESG).
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Detection: Absorbance at 360 nm, using uric acid formation (extinction coefficient: 6,220 M⁻¹cm⁻¹) .
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Unit Definition: 1 U = 1 µmol hypoxanthine generated per minute at 25°C .
Expression Platforms
E. coli-derived PNP is widely used due to high solubility and ease of purification .
Therapeutic and Industrial Applications
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Immunodeficiency Therapy: Enzyme replacement rescues T-cell apoptosis in PNP-deficient neurons .
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Biocatalysis: Synthesizes purine analogs like 2'-deoxyfluoro nucleosides for antiviral/anticancer drugs .
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Diagnostics: Coupled assays detect inorganic phosphate in biochemical reactions .
Research Findings
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Inhibitor Development: Transition-state analogues (e.g., Immucillin-H) achieve picomolar inhibition constants for T-cell cancer therapy .
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Neurological Defects: PNP deficiency induces mitochondrial apoptosis in neurons via p53 activation, reversible with exogenous PNP .
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Substrate Flexibility: Recombinant PNPs phosphorylate non-canonical substrates like 2'-amino-2'-deoxyadenosine .
