5UGF

Crystal structure of human purine nucleoside phosphorylase (F159Y) mutant complexed with DADMe-ImmG and phosphate

Summary for 5UGF

• Entry DOI: 10.2210/pdb5ugf/pdb
• Descriptor: Purine nucleoside phosphorylase, PHOSPHATE ION, 2-amino-7-{[(3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidin-1-yl]methyl}-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one, ... (4 entities in total)
• Functional Keywords: phosphorylase, inhibitor, transition state analogue, purine salvage pathway, transferase-transferase inhibitor complex, transferase/transferase inhibitor
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 6
• Total formula weight: 218819.84

Authors

Harijan, R.K.,Cameron, S.A.,Bonanno, J.B.,Almo, S.C.,Schramm, V.L. (deposition date: 2017-01-08, release date: 2017-06-21, Last modification date: 2023-10-04)

Primary citation

Harijan, R.K.,Zoi, I.,Antoniou, D.,Schwartz, S.D.,Schramm, V.L.
Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase.
Proc. Natl. Acad. Sci. U.S.A., 114:6456-6461, 2017

PubMed Abstract: Heavy-enzyme isotope effects (N-, C-, and H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (/ > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (/ < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage.

PubMed: 28584087
DOI: 10.1073/pnas.1704786114

Experimental method

X-RAY DIFFRACTION (2.2 Å)