Projects per year
Purine nucleoside phosphorylase (PNP) catalyzes phosphorolysis of 6-oxypurine nucleosides and deoxynucleosides. The transition state structure is oxacarbenium-like from kinetic isotope effects and transition state analogues (Immucillins) designed from this structure bind with pM affinity. Crystal structures have been solved with substrate, product and transition state analogues. The hypothesis emerging for catalysis is formation of an oxacarbenium ion transition state by neighboring group interactions from the 5'-hydroxyl of the ribosyl group and the enzyme-bound phosphate nucleophile. The catalytic site places neighbor oxygens the ribosyl 04', assisting electron contribution from the ribosyl group to the leaving group. This geometry supports an 'electronic promoting vibration' where protein groups fluctuate to bring oxygens closer, promoting electron expulsion. Computational chemistry dynamics (Schwartz, Project 4) will identify groups associated with this dynamic. Catalytic site mutations predicted to disrupt the promoting vibration will be made and tested. Isotope-edited infrared spectroscopy (Callender, Project 1) has established strong spectral bands associated with the phosphate nueleophile and the leaving group interactions. We propose time-resolved spectral analysis to correlate changes in protein dynamics, catalytic site chemistry, pH, leaving group and nucleophile interactions. T-jumps of dynamic equilibrium mixtures PNP with substrates and products will be induced by laser on a fast time scale followed by time-resolved monitoring of each parameter. Caged H+ will be used to initiate pH jumps to examine chemical and structural perturbations through proton donor/acceptor sites. Caged phosphate will be used to convert PNP.Immucillin to PNP.Immucillin.PO4, followed by isotope-edited following of the structural changes associated with slow-onset tight binding to resemble a transition state complex (Dyer, Project 3). Time-resolved spectra will be examined from psec to min time scales to follow local and global dynamics. Preliminary results establish rich IR spectral signatures for PO4 and leaving group interactions. These results will provide novel insights for the relationship between protein dynamics, ligand interactions and dynamics in catalysis.
|Effective start/end date||5/1/04 → 4/30/09|
- National Institute of General Medical Sciences: $211,972.00
- National Institute of General Medical Sciences: $126,958.00
- National Institute of General Medical Sciences: $123,489.00
- National Institute of General Medical Sciences: $130,022.00
- National Institute of General Medical Sciences: $210,855.00
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