Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleo side phosphorylase (PNP) was monitored by electrospray ionization mass spec trometry (ESI-MS) to probe protein conformational and dynamic changes induc ed by a substrate analogue, products. and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immuno deficiency, while B-cell immunity remains functional. Inhibitors of PNP hav e been proposed for treatment of T-cell leukemia, to suppress the graft-vs. -host response, or to counter type IV autoimmune diseases without destroyin g humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains w ith 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a K-d of 73 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no cataly tic site ligands no present. The substrate analogue and product prevented H /D exchange at 10 of the sites. Immucillin-H protected 32 protons from exch ange at full saturation. When one of the three subunits of the homotrimer i s filled with immucillin-H, and 27 protons are protected from exchange in a ll three subunits. Deuterium incorporation in peptides from residues 132-15 2 decreased in all complexes of PNP. The rate and/or extent of deuterium in corporation in peptides from residues 29-49. 50-70, 81-98, and 112-124 decr eased only in the complex with the transition state analogue. The peptide-s pecific H/D exchange demonstrates that (1) the enzyme is most compact in th e complex with immucillin-H, and (7) tilling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic si te, providing evidence for reduced protein dynamic motion caused by the tra nsition state analogue.