Catalytic Site Conformations in Human PNP by 19F-NMR and Crystallography

SummaryPurine nucleoside phosphorylase (PNP) is a target for leukemia, gout, and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate-bound enzyme and in complexes with catalytic site ligands, including transition state analogs. Chemical shift and line-shape changes associated with these complexes revealed dynamic coexistence of several conformational states in these regions in phosphate-bound enzyme and altered or single conformations in other complexes. These conformations were also characterized by X-ray crystallography. Specific 19F-Trp labels and X-ray crystallography provide multidimensional characterization of conformational states for free, catalytic, and inhibited complexes of human PNP.

► 19F-tryptophan insertions are NMR reporters for the catalytic site of human PNP ► Open and closed catalytic site conformations are faster than catalysis ► Catalytic site elements have multiple conformations until the site is filled ► Combined NMR and crystallography reveal dynamics and structure for PNP